Expression patterns of homeobox genes in the mouse vomeronasal organ at postnatal stages

Homeodomain proteins are encoded by homeobox genes and regulate development and differentiation in many neuronal systems. The mouse vomeronasal organ (VNO) generates in situ mature chemosensory neurons from stem cells. The roles of homeodomain proteins in neuronal differentiation in the VNO are poorly understood. Here we have characterized the expression patterns of 28 homeobox genes in the VNO of C57BL/6 mice at postnatal stages using multicolor fluorescent in situ hybridization. We identified 11 homeobox genes (Dlx3, Dlx4, Emx2, Lhx2, Meis1, Pbx3, Pknox2, Pou6f1, Tshz2, Zhx1, Zhx3) that were expressed exclusively in neurons; 4 homeobox genes (Pax6, Six1, Tgif1, Zfhx3) that were expressed in all non-neuronal cell populations, with Pax6, Six1 and Tgif1 also expressed in some neuronal progenitors and precursors; 12 homeobox genes (Adnp, Cux1, Dlx5, Dlx6, Meis2, Pbx2, Pknox1, Pou2f1, Satb1, Tshz1, Tshz3, Zhx2) with expression in both neuronal and non-neuronal cell populations; and one homeobox gene (Hopx) that was exclusively expressed in the non-sensory epithelium. We studied further in detail the expression of Emx2, Lhx2, Meis1, and Meis2. We found that expression of Emx2 and Lhx2 initiated between neuronal progenitor and neuronal precursor stages. As far as the sensory neurons of the VNO are concerned, Meis1 and Meis2 were only expressed in the apical layer, together with Gnai2, but not in the basal layer.     

Firing properties of accessory olfactory bulb mitral/tufted cells in response to urine delivered to the vomeronasal organ of gray short-tailed opossums

In comparison with many mammals, there is limited knowledge of the role of pheromones in conspecific communication in the gray short-tailed opossum. Here we report that mitral/tufted (M/T) cells of the accessory olfactory bulb (AOB) of male opossums responded to female urine but not to male urine with two distinct patterns: excitation followed by inhibition or inhibition. Either pattern could be mimicked by application of guanosine 5'-O-3-thiotriphosphate and blocked by guanosine 5'-O-2-thiodiphosphate, indicating that the response of neurons in this pathway is through a G-protein-coupled receptor mechanism. In addition, the inhibitor of phospholipase C (PLC), U73122, significantly blocked urine-induced responses. Male and female urine were ineffective as stimuli for M/T cells in the AOB of female opossums. These results indicate that urine of diestrous females contains a pheromone that directly stimulates vomeronasal neurons through activation of PLC by G-protein-coupled receptor mechanisms and that the response to urine is sexually dimorphic.     

Sex-specific responses to urinary chemicals by the mouse vomeronasal organ

Social behaviors of most mammals are affected by chemical signals, pheromones, exchanged between conspecifics. Previous experiments have shown that behavioral responses to the same pheromone differ depending on the sex and endocrine status of the respondent. Although the exact mechanism of this dimorphism is not known, one possible contributor may be due to sexually dimorphic receptors or due to differences in central processing within the brain. In order to investigate the differences in response between male and female mice to the same pheromonal stimulus two urinary compounds (2-heptanone and 2,5-dimethylpyrazine) were used to stimulate the production of Inositol (1,4,5)-trisphosphate (IP(3)) in microvillar membrane preparations of the vomeronasal organ as an indirect measurement of pheromonal stimulation. Incubation of such membranes from prepubertal mice with urine from the same sex or opposite sex, results in an increase in production of IP(3). This stimulation is mimicked by GTPgammaS and blocked by GDPbetaS. Furthermore we found that 2-heptanone present in both male and female urine was capable of stimulating increased production of IP(3) in the female VNO but not the male VNO. Finally, 2,5-dimethylpyrazine present only in female urine was also only capable of stimulating increased production of IP(3) in the female VNO.     

Seasonal morphometry of the vomeronasal organ in the marsupial mouse,Antechinus subtropicus

The vomeronasal system consists of a peripheral organ and the connected central neuronal networks. The central connections are sexually dimorphic in rodents, and in some species, parameters of the vomeronasal organ (VNO) vary with sex, hormonal exposure, body size and seasonality. The VNO of the dasyurid marsupial mouse, Antechinus subtropicus is presumed to be functional. The unusual life history (male semelparity) is marked by distinct seasonality with differences in hormonal environments both between males and females, and in males at different time points. Body size parameters (e.g., length, weight) display sexual dimorphism and, in males, a pronounced weight gain before breeding is followed by a rapid decline during the single, short reproductive season. VNO morphometry was investigated in male and female A. subtropicus to identify possible life cycle associated activity. The overall length of the VNO is positively correlated with the size of the animal. The amount of sensory epithelium exhibits a negative correlation, decreasing with increasing size of the animal. The effects of sex and breeding condition are not obvious, although they do suggest that sensory vomeronasal epithelium mass declines in the breeding period. The VNO may be more important in A. subtropicus before breeding when it may participate in synchronising reproduction and in the development of the male stress response. J. Morphol. 277:1517–1530, 2016. © 2016 Wiley Periodicals, Inc.     

Chiropteran vomeronasal complex and the interfamilial relationships of bats

Within the extant orders of living mammals, the distribution of the vomeronasal organ (VNO) and associated structures is very stable, being universally present in the vast majority or universally absent in cetaceans and sirenians. Chiroptera is the most noteworthy exception, with variation in the absence or presence of the vomeronasal complex occurring even at the species level in some instances. The VNO and/or its component structures, such as the accessory olfactory bulb, were studied in serially sectioned snouts and brains from 114 genera and 292 species representing all extant chiropteran families except Myzopodidae and Antrozoidae. Taxa were scored for the following characters: (1) degree of formation of the vomeronasal epithelial tube, (2) shape of the vomeronasal cartilage, (3) occurrence of the nasopalatine duct, and (4) occurrence of the accessory olfactory bulb. To reconstruct the evolutionary history of the bat vomeronasal complex, the distributions of these four characters were mapped, using the computer program MacClade, onto chiropteran phylogenies in the literature derived from other data sets. In all phylogenies, these four characters exhibit a high degree of homoplasy, only part of which is accounted for by several polymorphic taxa. However, perhaps the most remarkable result is that in the most parsimonious solutions the absence of the vomeronasal epithelial tube and accessory olfactory bulb is identified as primitive for Chiroptera, with both structures reevolving numerous times: such a scenario would be unique to bats among mammals. An alternative, though less parsimonious interpretation, which does not require reevolution of this very complex system, is that a well-developed vomeronasal epithelial tube is primitive for Chiroptera, as in nearly all other orders of mammals, but has been reduced or lost in the majority of families. Explication of the peculiar evolutionary history of the vomeronasal system in bats awaits studies on the adult morphology in the more than 630 species not yet examined and, in particular, on ontogeny, which to date is known for only a handful of taxa.A preliminary account of this research was presented at the Tenth International Bat Research Conference and Twenty-Fifth North American Bat Research Symposium held at Boston University, Massachusetts, on 6–11 August 1995.     

Resolution and sensitivity of high field nuclear magnetic resonance spectroscopy

NMR frequency assignments are usually considered a prerequisite for the analysis of NOESY spectra, in turn required for the calculation of biomolecular structures. In contrast, as we propose here, relatively high numbers of unambiguous NOE identities can be consistently achieved in an automated manner by relying only on grouping resonances into connected spin systems. To achieve this goal, we have developed for proteins two protocols, SPI and BACUS, based on Bayesian inference. SPI (Grishaev and Llinás, 2002c) produces a list of the (1)H resonance frequencies from homo- and hetero-nuclear multidimensional spectra, grouped into effective spin systems. BACUS automatically establishes probabilistic identities of NOESY cross-peaks in terms of the chemical shifts provided by SPI. BACUS requires neither assignment of resonances nor an initial structural model. It successfully copes with chemical shift overlap and does so without cycling through 3D structure calculations. The method exploits the self-consistency of the NOESY graph by taking advantage of a network of J- as well as NOE-connected "reporter" protons sorted via SPI. BACUS was validated by tests on experimental NOESY data recorded for the col 2 and kringle 2 domains.     

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.     

Reinforcement selection acting on the European house mouse hybrid zone

Behavioural isolation may lead to complete speciation when partial postzygotic isolation acts in the presence of divergent‐specific mate‐recognition systems. These conditions exist where Mus musculus musculus and M. m. domesticus come into contact and hybridize. We studied two mate‐recognition signal systems, based on urinary and salivary proteins, across a Central European portion of the mouse hybrid zone. Introgression of the genomic regions responsible for these signals: the major urinary proteins (MUPs) and androgen binding proteins (ABPs), respectively, was compared to introgression at loci assumed to be nearly neutral and those under selection against hybridization. The preference of individuals taken from across the zone regarding these signals was measured in Y mazes, and we develop a model for the analysis of the transition of such traits under reinforcement selection. The strongest assortative preferences were found in males for urine and females for ABP. Clinal analyses confirm nearly neutral introgression of an Abp locus and two loci closely linked to the Abp gene cluster, whereas two markers flanking the Mup gene region reveal unexpected introgression. Geographic change in the preference traits matches our reinforcement selection model significantly better than standard cline models. Our study confirms that behavioural barriers are important components of reproductive isolation between the house mouse subspecies.     

Convergent vomeronasal system reduction in mammals coincides with convergent losses of calcium signalling and odorant‐degrading genes

The vomeronasal system (VNS) serves crucial functions for detecting olfactory clues often related to social and sexual behaviour. Intriguingly, two of the main components of the VNS, the vomeronasal organ (VNO) and the accessory olfactory bulb, are regressed in aquatic mammals, several bats and primates, likely due to adaptations to different ecological niches. To detect genomic changes that are associated with the convergent reduction of the VNS, we performed the first systematic screen for convergently inactivated protein‐coding genes associated with convergent VNS reduction, considering 106 mammalian genomes. Extending previous studies, our results support that Trpc2, a cation channel that is important for calcium signalling in the VNO, is a predictive molecular marker for the presence of a VNS. Our screen also detected the convergent inactivation of the calcium‐binding protein S100z, the aldehyde oxidase Aox2 that is involved in odorant degradation, and the uncharacterized Mslnl gene that is expressed in the VNO and olfactory epithelium. Furthermore, we found that Trpc2 and S100z or Aox2 are also inactivated in otters and Phocid seals for which no morphological data about the VNS are available yet. This predicts a VNS reduction in these semi‐aquatic mammals. By examining the genomes of 115 species in total, our study provides a detailed picture of how the convergent reduction of the VNS coincides with gene inactivation in placental mammals. These inactivated genes provide experimental targets for studying the evolution and biological significance of the olfactory system under different environmental conditions.     

Maturation of vomeronasal receptor neurons in vitro by coculture with accessory olfactory bulb neurons

To analyze the mechanisms of perception and processing of pheromonal signals in vitro, we previously developed a new culture system for vomeronasal receptor neurons (VRNs), referred to as the vomeronasal pocket (VN pocket). However, very few VRNs were found to express the olfactory marker protein (OMP) and to have protruding microvilli in VN pockets, indicating that these VRNs are immature and that VN pockets are not appropriate for pheromonal recognition. To induce VRN maturation in VN pockets, we here attempted to coculture VN pockets with a VRN target-accessory olfactory bulb (AOB) neurons. At 3 weeks of coculture with AOB neurons, the number of OMP-immunopositive VRNs increased. By electron microscopy, the development of microvilli in VRNs was found to occur coincidentally with OMP expression in vitro. These results indicate that VRN maturation is induced by coculture with AOB neurons. The OMP expression of VRNs was induced not only by AOB neurons but also by neurons of other parts of the central nervous system (CNS). Thus, VRN maturation requires only CNS neurons. Since the maturation of VRNs was not induced in one-well separate cultures, the nonspecific induction of OMP expression by CNS neurons suggests the involvement of a direct contact effect with CNS in VRN maturation.     

The septal organ of the rat during postnatal development

The septal organ of Masera (SO) is a small, isolated patch of olfactory epithelium, located in the ventral part of the nasal septum. We investigated in this systematic study the postnatal development of the SO in histological sections of rats at various ages from the day of birth (P1) to P666. The SO-area increases to a maximum at P66-P105, just as the animals reach sexual maturity, and decreases thereafter, significantly however only in males, indicating a limited neurogenetic capacity for regeneration. In contrast, the main olfactory epithelium area continues to expand beyond P300. The modified respiratory epithelium ('zwischen epithelium') separating the SO and the main olfactory epithelium contains a few olfactory neurons up to age P66. Its length increases postnatally so that the SO becomes more ventral to the OE. Although the position of the SO relative to other anatomical landmarks changes with development it is consistently located just posterior to the opening of the nasopalatine duct (NPAL). Thus, a possible function of the SO is in sensing chemicals in fluids entering the mouth by licking and then delivered to the nasal cavity via the NPAL; therefore the SO may be involved in social/sexual behavior as is the vomeronasal organ (VNO). We suggest that the SO is a separate accessory olfactory organ with properties somewhat different from both OE and VNO and may exist only in species where the NPAL does not open into the VNO.     

High-resolution proton nuclear magnetic resonance spectroscopy of chloride peroxidase: identification of new forms of the enzyme

Chloride peroxidase from the mold Caldariomyces fumago in the native high-spin iron(III) and low-spin cyanoiron (III) states has been subjected to high-field proton nuclear magnetic resonance spectroscopic measurements. Signals shifted well outside the diamagnetic envelope by the paramagnetic iron(III) center are surprisingly insensitive to pH changes over the range from pH 3 to pH 7. The previously identified major form of chloride peroxidase (form A) and the minor form (B) show very similar chemical shift patterns. Of greatest significance, however, is the discovery that each of the separable forms of the enzyme exhibits splitting of porphyrin ring methyl resonances. The appearance of two sets of signals in both native and cyanide-complexed enzyme is best explained by the existence of two additional forms of the A and B isoenzymes. Structural differences for the newly identified forms of chloride peroxidase must be located in the vicinity of the heme prosthetic group.     

Differential development of odorant receptor expression patterns in the olfactory epithelium: a quantitative analysis in the mouse septal organ

The rodent olfactory epithelium expresses more than 1000 odorant receptors (ORs) with distinct patterns, yet it is unclear how such patterns are established during development. In the current study, we investigated development of the expression patterns of different ORs in the septal organ, a small patch of olfactory epithelium predominantly expressing nine identified ORs. The presumptive septal organ first appears at about embryonic day 16 (E16) and it completely separates from the main olfactory epithelium (MOE) at about postnatal day 7 (P7). Using in situ hybridization, we quantified the densities of the septal organ neurons labeled by specific RNA probes of the nine abundant OR genes from E16 to postnatal 3 months. The results indicate that olfactory sensory neurons (OSNs) expressing different ORs have asynchronous temporal onsets. For instance, MOR256-17 and MOR236-1 cells are present in the septal organ at E16; however, MOR0-2 cells do not appear until P0. In addition, OSNs expressing different ORs show distinct developmental courses and reach their maximum densities at different stages ranging from E16 (e.g. MOR256-17) to 1 month (e.g. MOR256-3 and MOR235-1). Furthermore, early onset does not correlate with high abundance in adult. This study reveals a dynamic composition of the OSNs expressing different ORs in the developing olfactory epithelium.     

High-resolution phosphorus nuclear magnetic resonance spectroscopy of transfer ribonucleic acids: multiple conformations in the anticodon loop

The temperature dependence of the 31P NMR spectra of yeast phenylalanine tRNA, E. coli tyrosine, glutamate (2), and formylmethionine tRNA is presented. The major difference between the 31P NMR spectra of the different acceptor tRNAs is in the main cluster region between -0.5 and -1.3 ppm. This confirms an earlier assignment of the main cluster region to the undistorted phosphate diesters in the hairpin loops and helical stems. In addition the 31P NMR spectra for all tRNAs reveal approximately 16 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester. In the presence of 10 mM Mg2+ most scattered and main cluster signals do not shift between 22 and 66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles. At greater than 70 degrees C, all of the signals merge into a single random-coil conformation signal. A number of the scattered peaks are shifted (0.2-1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ and spermine as a result of a conformational transition in the anticodon loop. The 31P NMR spectrum of the dimer formed between yeast tRNAPhe and E. coli tRNA 2Glu is reported. This dimer simulates codon-anticodon interaction since the anticodon triplets of the two tRNAs are complementary. Evidence is presented that the anticodon-anticodon interaction alters the anticodon conformation and partially disrupts the tertiary structure of the tRNA.     

Genetically determined electrophoretic variants of the major urinary protein (Mup) complex in mouse urine

The major urinary protein (Mup-complex) excreted in mouse urine, has been studied electrophoretically both on starch gel and on cellogel.
On stargel six anodally migrating protein bands were observed. These bands are designated component 3, 2', 2, 1, and 4 (i.e. two bands) in the order of decreasing mobility toward the anode. The slower protein band of component 4 on starch gel was not observed on cellogel.
By testing mouse inbred strains, we were able to dinstinguish five male and four female Mup phenotypes. Test crosses suggested a four-allelic ( a, b, c, d, ) variation with regard to components 2', 2 and 1: 'group A' strains showed component 1, 'group B' strains components 1 and 2, 'group C' and 'group F strains none, and 'group D' strains showed components 1 and 2'. Component 3 may be encoded by another Mup locus, although no crossing-over has been observed: presence (A, B, D, and F strains), absence (C strains). Insufficiently reproducible demonstration of the variation with regard to component 4, forced us to exclude this component for strain distinction.
The Mup phenotypes described, can be useful for the detection of certain strain contaminations, especially if F₁ hybrid Mup phenotypes are distinguishable     

In vivo phenotyping of the ob/ob mouse by magnetic resonance imaging and 1H-magnetic resonance spectroscopy

Objective: We studied ob/ob and wild‐type (WT) mice to characterize the adipose tissues depots and other visceral organs and to establish an experimental paradigm for in vivo phenotyping. Research Methods and Procedures: An in vivo evaluation was conducted using magnetic resonance imaging and ¹H‐magnetic resonance spectroscopy (¹H‐MRS). We used T1‐weighted images and three‐dimensional spin echo T1‐weighted images for the morphological analysis and ¹H‐MRS spectra on all body mass, as well as ¹H‐MRS spectra focalized on specific lipid depots [triglyceride (TG) depots] for a molecular analysis. Results: In ob/ob mice, three‐dimensional evaluation of the trunk revealed that ～64% of the volume consists of white adipose tissue, which is 72% subcutaneous and 28% visceral. In vivo ¹H‐MRS showed that 20.00 ± 6.92% in the WT group and 58.67 ± 6.65% in the ob/ob group of the total proton content is composed of TG protons. In in vivo‐localized spectra of ob/ob mice, we found a polyunsaturation degree of 0.5247 in subcutaneous depots. In the liver, we observed that 48.7% of the proton signal is due to water, whereas in the WT group, the water signal amounted to 82.8% of the total proton signal. With the sequences used, the TG amount was not detectable in the brain or kidneys. Discussion: The present study shows that several parameters can be obtained by in vivo examination of ob/ob mice by magnetic resonance imaging and ¹H‐MRS and that the accumulated white adipose tissue displays low polyunsaturation degree and low hydrolipidic ratio. Relevant anatomical alterations observed in urinary and digestive apparatuses should be considered when ob/ob mice are used in experimental paradigms.     

High-resolution nuclear magnetic resonance spectroscopy studies of polysaccharides crosslinked by sodium trimetaphosphate: a proposal for the reaction mechanism

An NMR spectroscopy study ((31)P, (1)H, (13)C) of the postulated crosslinking mechanism of sodium trimetaphosphate (STMP) on polysaccharides is reported using methyl alpha-D-glucopyranoside as a model. In a first step, reaction of STMP with Glc-OMe gives grafted sodium tripolyphosphate (STPP(g)). On the one hand, STTP(g) can react with a second alcohol functionality to give a crosslinked monophosphate. On the other hand, a monophosphate (grafted phosphate) could be obtained by alkaline degradation of STPP(g). NMR spectroscopy allows to detect the various species formed and to obtain the crosslinking density of STMP-polysaccharides hydrogels.     

High resolution X-ray structures of mouse major urinary protein nasal isoform in complex with pheromones

In mice, the major urinary proteins (MUP) play a key role in pheromonal communication by binding and transporting semiochemicals. MUP‐IV is the only isoform known to be expressed in the vomeronasal mucosa. In comparison with the MUP isoforms that are abundantly excreted in the urine, MUP‐IV is highly specific for the male mouse pheromone 2‐sec‐butyl‐4,5‐dihydrothiazole (SBT). To examine the structural basis of this ligand preference, we determined the X‐ray crystal structure of MUP‐IV bound to three mouse pheromones: SBT, 2,5‐dimethylpyrazine, and 2‐heptanone. We also obtained the structure of MUP‐IV with 2‐ethylhexanol bound in the cavity. These four structures show that relative to the major excreted MUP isoforms, three amino acid substitutions within the binding calyx impact ligand coordination. The F103 for A along with F54 for L result in a smaller cavity, potentially creating a more closely packed environment for the ligand. The E118 for G substitution introduces a charged group into a hydrophobic environment. The sidechain of E118 is observed to hydrogen bond to polar groups on all four ligands with nearly the same geometry as seen for the water‐mediated hydrogen bond network in the MUP‐I and MUP‐II crystal structures. These differences in cavity size and interactions between the protein and ligand are likely to contribute to the observed specificity of MUP‐IV.