Collectively, these experiments demonstrated that signals required for pup-directed attacks can be reconstituted using inanimate cues. Importantly, specific chemical cues produced in pup salivary glands significantly enhanced pup-directed aggression, while cues known to be involved in adult-adult aggression, such as adult male urine, did not.

Interestingly, dummies coated with PBS alone elicited some low level of attacks ( Figure 1 E). However, swabbing dummies with pup salivary extracts significantly increased the duration of aggressive bouts and chemoinvestigative contacts compared to PBS- or adult-male-urine-swabbed dummies ( Figure 1 F). The initial 3 min of the behavior assay contained the highest frequency of aggressive events, and salivary extracts led to significantly higher attack duration and contact time in this period compared to PBS- or urine-swabbed dummies ( Figure 1 G).

To further characterize the minimal set of stimuli required for pup-directed aggression, we swabbed dummies with pup salivary extracts as a source of pup cues; phosphate buffered saline (PBS) as negative control; and adult male urine, a source of chemosignals known to trigger male-directed attacks ().

Pup dummies conditioned for an hour with scents from a cage containing a mother and pups elicited robust attacking behavior ( Video S1 ). Intriguingly, the majority of males attacking pup dummies displayed the stereotypical behaviors seen in aggressive episodes with live or dead pups, such as chemo-investigation, rough handling, aggressive grooming, and biting. We compared aggression directed against dead or dummy pups swabbed with pup salivary gland extracts and established that dummies conditioned with pup scents (e.g., pup salivary extracts) provoked naturalistic pup-directed aggression ( Figure 1 B; Videos S1 and S2 ), such that the initial investigation of pups by males switches to quickly alternating chemo-investigation and biting. Occasionally, males grab dummies by the mouth and carry them within the cage with jerky movements, before repeatedly biting them on the ground ( Videos S1 and S2 ). Compared to dummies swabbed with pup scents, dead pups provoked more sustained attacks and shorter latency to the first attack ( Figures 1 B–1D), suggesting that additional cues emitted by dead pups intensify male aggression. However, the aggressive behavior of males toward silicone dummies conditioned with pup scents closely mimics attacks of real pups.

Virgin males attack dead pups vigorously, indicating that vocalization, body temperature, and movement are not essential cues for pup-directed aggression ( Figures 1 A and 1B ). Further, pup-directed male aggression occurs in full darkness, suggesting that vision is also dispensable (not shown). This prompted us to design silicone pup dummies recapitulating the morphological traits of a mouse pup, such as its size, shape, and texture ( Figure 1 A).

(I) p values of the paired t test for the differences in aggressive bout durations between decoys of various shapes swabbed with PBS and pup salivary extracts (blue) or between decoys swabbed with male urine and pup salivary extracts (brown) tested in 2 min sliding windows. Note the late attack against the “hybrid” decoy. N = 15.

(H) Raster plots of behaviors by virgin males exposed to various silicone dummies. Each event marks an interaction between males and the introduced pup decoy swabbed with pup salivary extract, with events categorized as aggressive (red) or non-aggressive (blue). N = 15.

(G) Sliding window analysis of aggressive interactions between males and dummies swabbed with various chemical cues. Top graph shows the cumulative duration of aggressive bouts in 2 min sliding windows. Bottom graph shows the p values of the paired t test for differences in aggressive bout durations between dummies swabbed with PBS and pup salivary extracts (blue) or between dummies swabbed with male urine and pup salivary extracts (brown) in 2 min sliding windows. N = 15.

(E and F) Durations of aggressive interactions (E) and all contacts (F) in the initial 3 min of encounters in which males are presented with dummies swabbed with PBS (blue), pup salivary extracts (red), and adult male urine (yellow). ∗ p < 0.05, ∗∗ p < 0.01 by paired t test, N = 15. Connected circles represent data obtained with the same males.

(C) Total aggression per 5 min assay is significantly longer when males are exposed to dead pups compared to dummies (p < 0.001 by t test). N = 19.

These results revealed that the identification of pups by aggressive males is a multi-sensory process, involving the recognition of specific physical and chemical signals.

Fast-frame videography of adult males investigating pups indicated that males made extensive orofacial contact with pups ( Video S3 ), prompting us to examine the effect of infant morphological traits in dummy-directed aggression. We tested three additional dummy shapes: the brick, an unnatural object of similar size with sharp edges; the blob, a legless dummy with pup-like head and body curvature; and the hybrid, which combined the brick with limbs and a tail ( Figure 1 A). Exposure of males to a brick failed to induce aggression, even when painted with pup salivary extracts (p < 0.001, Student’s t test, N = 15; Figures 1 H–1J and S1 ). Likewise, the blob did not elicit aggression (p = 0.12, Student’s t test, N = 15). Strikingly, however, the hybrid shape with hind and front legs and tail partially restored aggression (p < 0.01, Student’s t test, N = 15), although to a smaller extent than the more realistic dummy shape (p < 0.05, Student’s t test, N = 15; Figure 1 J).

(A–D) Raster plots of behavioral events by virgin males exposed to various types of silicone shapes swabbed with pup salivary extract, PBS, or male urine. The aggressive and non-aggressive bouts are marked by red and blue boxes, respectively. Below each raster plot is a sliding window analysis (2 min), using the same procedure as in Figure 1 I. N = 15.

Surprisingly, published work as well as our data revealed that none of the identified VRs were specific to pup cues ( Figure 2 J), such that they were each also stimulated by either adult male or female cues ( Figures 2 J and S2 ). Thus, none of the receptors activated by pup cues conveyed specific information about infants, although they may do so as a group.

(B) Response of Vmn2r88+ neurons to pups in virgin females exhibiting maternal behaviors. 78.9 ± 6.2% of Vmn2r88+ neurons overlap with Egr1 (245 Vmn2r88+ neurons examined, N = 3 animals). Arrow marks the overlap of Egr1 and VR signals.

(A) Egr1 is robustly induced in VNO neurons expressing Vmn2r122/123 in virgin males exposed to male C57BL/6J submaxillary gland extracts (82.2 ± 6.0% of Vmn2r122/123 cells are Egr1 + ; Error in SEM; 38 Vmn2r122/123 + neurons from 2 animals examined).

Among these transcripts were genes encoding VRs, but no genes encoding odorant receptors or other classes of known chemosensory receptors ( Figure 2 F), as well as V1rc1, V1rc30, Vmn2r65, and Vmn2r88, i.e., four out of five VR transcripts identified in our earlier screen ( Figure 2 G). Two closely related genes V1rc1 and V1rc30, which could not be distinguished from each other by in situ hybridization, encoded receptors that are both activated by pup cues. In addition, a VR sequence annotated as pseudogene Vmn2r-ps159 emerged from the pS6 screen. Upon closer examination, these reads matched perfectly with the sequences of Vmn2r122 and Vmn2r123, previously cloned but not mapped to the genome (). We confirmed that VNO neurons expressing Vmn2r122 or 123 (denoted as Vmn2r122/123 since they could not be distinguished by in situ hybridization) were robustly activated by pup cues, accounting for 14.0% ± 2.2% of Egr1neurons ( Figure 2 H). Overall, the expression of the seven VR genes identified here accounted for 92.2% ± 1.2% of the VNO neurons activated by pups ( Figure 2 I), suggesting that we reached a nearly comprehensive identification of VRs to infant cues.

Approximately 20% of Egr1cells were not covered by the pool of five VR probes, suggesting that one or few additional VRs to pup cues may exist that were not identified in this candidate-based screen ( Figure 2 C bottom, green arrows). We therefore performed a second, unbiased, screen of transcripts associated with the phosphorylated ribosomal protein S6 (pS6) in activated neurons (). Control experiments confirmed that the phosphorylation of the S6 is robustly induced in Egr1neurons upon pup exposure ( Figure 2 D). Next, we captured the transcriptome of neurons activated by pup cues or fresh bedding by immunopurification of phospho-ribosome-associated RNAs ( Figure 2 E). Data from RNA sequencing (RNA-seq) of pS6 pulled-down transcripts were analyzed and searched for transcripts enriched in VNOs from animals exposed to pup cues.

Next, we used an in situ hybridization-based screen to uncover the identity of VRs expressed in Egr1neurons following pup encounters by aggressive males ( STAR Methods ). Five VRs were identified: Vmn2r88 (representing 43.4% ± 0.8% of Egr1cells; mean ± SEM), V1rc1 or V1rc30 (15.6% ± 1.5%; our probe does not distinguish between these two closely related transcripts), Vmn2r65 (11.2% ± 1.6%), and V1ri9 (9.0% ± 1.7%) ( Figures 2 C and 2H).

Following exposure to pups, significantly fewer Egr1cells were detected in the VNO of virgin females and non-aggressive virgin males compared to aggressive virgin males (p < 0.001 by Student’s t test, virgin females N = 9, non-aggressive males N = 3; Figure 2 B). This is consistent with a previous report showing that VNOs of fathers have reduced c-fos activation following pup exposure compared to virgin males (), and it extends this observation to males lacking aggressive display toward pups, irrespective of their status as virgin or fathers.

Because pups are taken from the mother’s cage and transferred to the behavioral arena, we tested the potential contribution of maternal odor in our assays and sampled bedding from cages in which mothers had been separated from their pups and housed alone for 2 days. This exposure resulted in the activation of about 4-fold fewer Egr1neurons than from pup exposure ( Figures 2 A and 2B), suggesting that our assay robustly monitors VNO activation by pup cues, even in the context of mother-infant cohabitation.

We next aimed to analyze in more detail the contribution of VNO detection in this behavior. To search for VRs activated by pups, we assessed the induction of the immediate early gene Egr1, a sensitive molecular readout of VNO neural activity (), and visualized the activation of subpopulations of VNO neurons following encounters of adult virgin males and females with C57BL/6J pups ( Figures 2 A and 2B ).

(J) Summary of responses by VRs, identified based on their recognition of pup cues, to non-pup cues, from data byand data shown in Figure S2 A.

(H) Comprehensive set of VRs activated by pup cues. Double FISH with individual VR and Egr1 probes are shown the percentages of co-labeled neurons (N = 3 animals with total number of VR + cells analyzed indicated in parentheses).

(G) List of VRs identified in the pS6-based screen. The screen failed to identify the sparsely represented V1ri9 identified by the candidate VR screen (shown in C).

(F) MA (log 2 difference over signal strength) plot showing the enrichment of specific VR in the VNOs of virgin males exposed to pup stimuli compared to fresh bedding. Red dots represent genes significantly (p < 0.1) over-represented in pup-exposed VNO samples. Green dots represent enriched VR genes.

(D) Immunofluorescence with anti-pS6 antibody and in situ hybridization with RNA probe to Egr1 demonstrates co-expression (arrows) in VNO neurons activated by pup cues (94.7% ± 2.2% of Egr1 + neurons are pS6 + ; mean ± SEM; 699 Egr1 + cells from three animals examined).

(C) Top: Double fluorescence in situ hybridization (FISH) with RNA probes to Egr1 and candidate VRs led to the identification of five VRs responding to pup cues. Arrows indicate cells in which Egr1 (green) and VR signals (red) co-localize. Bottom: Double FISH with RNA probes to Egr1 (green) and a mix of the five VR probes shown in panels above. White arrows point to co-labeled neurons, and green arrows indicate Egr1 + neurons that express none of the five identified VRs.

(A and B) Egr1 induction in the VNO of virgin male and female mice upon pup exposure (A). The number of Egr1 + VNO neurons identified in these experiments is shown in (B). Each data point represents one animal. Number of animals used is indicated in the graph. ∗∗∗ p < 0.001 by t test. Dark lines indicate the mean, and light lines indicate the quartiles.

Thus, both adults and pups produce ligands for Vmn2r65 and Vmn2r88, further strengthening our initial observation that receptors identified based on their activation by pup cues also recognize adult cues. Moreover, our data suggest that responses of Vmn2r65-, 88-, and Vmn2r122/123-expressing neurons to pup cues are neither sex nor strain specific.

Interestingly, Vmn2r65-expressing neurons were exclusively stimulated by extracts of adult female submaxillary and sublingual glands but not by extracts from any other tissues nor by female urine ( Figures 3 B and 3C). Moreover, this activity was exclusively present in female but not male tissues ( Figure 3 F). In contrast, the ligand activity for Vmn2r88 was detected in extracts from multiple tissues in both males and females, most strongly in salivary and lacrimal glands ( Figures 3 B, 3D, and 3F). Salivary extracts from C57BL/6J pup also activated Vmn2r65- and Vmn2r88-expressing neurons ( Figures 3 B and 3E). In addition, salivary extracts from both male and female pups from four distinct mouse strains stimulated Vmn2r65-, Vmn2r88-, and Vmn2r122/123-expressing neurons ( Figure S3 ).

(E) Quantification of in situ hybridization signals. The percentage of VR + neurons that also co-express Egr1 was quantified in the above experiments. The errors are in SEM, and total numbers of VR + neurons quantified are indicated in parentheses.

(A–D) In situ hybridization with probes of Egr1 and vomeronasal receptors on VNOs of virgin CD-1 males exposed to salivary gland extracts of male or female pups from 4 different strains (n = 2 animals per stimulus).

Since Vmn2r65 and Vmn2r88 were robustly activated by both adult and pups cues, we first tested the ability of tissue homogenates from organs previously implicated in pheromone production and release, i.e., adult salivary and lacrimal glands ( Figure 3 A), as well as that of urine, to stimulate Vmn2r65- and Vmn2r88-expressing VNO neurons.

(E and F) Vmn2r88- and Vmn2r65-ligand activities are found in both pup (E) and adult tissue (F) extracts. All experiments were performed on two animals per stimulus, and the mean percentage of VR + Egr1 + neurons in VR + cells in virgin male VNOs is indicated, along with the number of cells analyzed.

(D) Vmn2r88-ligand activity is found in extracts of multiple glands. RNA FISH with Egr1 and Vmn2r88 probes on VNOs from virgin males after exposure to urine and gland extracts from adult virgin males. Arrows mark Egr1 and Vmn2r88 co-expression.

(C) Vmn2r65-ligand activity is found exclusively in female submaxillary gland extract. RNA FISH with Egr1 and Vmn2r65 probes on virgin male VNOs after exposure to urine and gland extracts from adult virgin females. Arrows mark Egr1 and Vmn2r65 co-expression.

Next, we sought to exploit the identification of VRs activated by pups to search for the corresponding molecular cues. Over 50% of VNO neurons activated by pups express V2Rs, among which the majority express either Vmn2r65 or Vmn2r88, prompting us to focus on the specific compounds recognized by these two VRs.

This strategy provided us with short lists of candidate ligands for Vmn2r65 and Vmn2r88, which were further narrowed down based on our earlier identification of the strain, sex, and salivary gland specificity of ligand activities ( Figures S4 A and 5 A). Control homogenates from different tissues, strains (C57BL/6J and 129S1/SvImJ), and sexes were subjected to identical purification procedures. Specifically, fractionation from male salivary glands was used as a negative control for the Vmn2r65-stimulating activity, which we had earlier identified in gland extracts from pups and adult females only. Likewise, salivary gland extracts from adult males of the 129S1/SvImJ strain had no or only very weak activity on neurons expressing Vmn2r88 and thus served as a negative control for this activity. As further described below, the combination of Egr1 induction to monitor ligand activity on specific VNO neurons, together with our MS-based screening provided a powerful and efficient platform to identify novel pheromone ligands.

We performed biochemical fractionations of tissue homogenates from adult C57BL/6J salivary glands to isolate compounds stimulating Vmn2r65- and Vmn2r88-expressing neurons ( Figures 4 A and 5 A ). First, we used size exclusion chromatography to separate molecules by size then exposed animals to individual fractions and quantified the percentage of activated (Egr1) cells among Vmn2r65- or Vmn2r88-expressing VNO neurons. Our initial analysis revealed that ligands for Vmn2r65 and Vmn2r88 were likely macromolecules with molecular weight of 60–80 kilodaltons (kDa) and 30–40 kDa, respectively ( Figures 4 B and 5 B). We further purified these samples by ion exchange chromatography ( Figures 4 C, 4D, 5 D, and 5E) and analyzed active fractions by mass spectrometry (MS; Method Details ).

(N) The boxplot shows the quantification of the Vmn2r88-expressing neurons co-labeled with Egr1. Four sections from two animals were quantified for each time point.

(M) Recombinant Hbb-bt-Hba-a1 was stored at room temperature for indicated period of time and tested for its ability to stimulate Vmn2r88-expressing neurons. Two CD-1 virgin males were used for this test per sample.

(L) Hemoglobin is readily detectable in the cage of pregnant and post-partum females with pups. Shown is a protein blot of bedding extracts probed by anti-Hb beta antibody. Bedding from three females (A, B, C) for each condition and males (D, E, F) are used.

(K) Plot of Egr1 signals (represented as fold increase from the standard deviation of image background) in Vmn2r88-expressing neurons, stimulated by bedding of cross-fostered cages. Bedding from cross-fostered cages (one 129S1/SvImJ mother with C57BL/6J pups, one 129S1/SvImJ mother with 129S1/SvImJ pups, one C57BL/6J mother with 129S1/SvImJ pups, N = 3 each). ∗∗ p < 0.01 by Student’s t test.

(J) Vmn2r88-ligand activity is deposited in bedding of males and in bedding of mothers cohabitating with infants. Mother and pup bedding each robustly excites Vmn2r88-expressing neurons, as evidenced by RNA FISH showing the colocalization of Egr1 and Vmn2r88 signals in the VNO of animals exposed to these cues (arrows). All experiments used three or four animals, and the percentage of Vmn2r88 + Egr1 + cells in Vmn2r88 + neurons are indicated, along with the total number of cells analyzed. All errors are in SEM.

(H and I) Specificity of Vmn2r88 activation by Hbs. Virgin males were exposed to recombinant Hbb-b1-Hba-a1 and Hbb-b2-Hba-a1, and their VNOs were analyzed by RNA FISH with Egr1 and Vmn2r88 probes. The intensities of the Egr1 signals in Vmn2r88-expressing neurons in (G) and (H), represented as fold increase from the standard deviation of image background, are plotted in a graph in (I). Each dot represents one cell. ∗∗∗ p < 0.001 by t test.

(G) Recombinant Hbb-bt-Hba-a1 robustly activates Vmn2r88-expressing neurons (94.1% ± 0.9% of Vmn2r88-expressing neurons; mean ± SEM; 240 Vmn2r88-expressing neurons from four animals examined) and Vmn2r122/123-expressing neurons (63.4% ± 16.4% of Vmn2r122/123-expressing neurons; 184 Vmn2r122/123-expressing neurons, three animals). Virgin males were exposed to recombinant Hbs, and their VNOs were analyzed for the activation of Egr1 in Vmn2r88- or Vmn2r122/123-expressing neurons.

(C) Adult C57BL/6J blood activates Vmn2r88-expressing VNO neurons (male blood: 92.5% ± 1.8% of Vmn2r88-expressing neurons; error in SEM; 161 Vmn2r88-expressing neurons from two animals; female blood: 97.3% ± 0.4% of Vmn2r88-expressing neurons; 152 Vmn2r88-expressing neurons from two animals examined).

(B) Activity profile of C57BL/6J male salivary gland extracts after gel filtration chromatography. The activity was assessed as the percentage of Vmn2r88-expressing neurons with Egr1 signal.

(F) Exposure of virgin males to recombinant Smgc results in specific activation of Vmn2r65 + VNO neurons. RNA FISH of VNO sections from virgin males exposed to recombinant Smgc shows co-localization of Egr1 and Vmn2r65 (arrows) (84.2% ± 5.0% of Vmn2r65 + neurons; error in SEM; 166 Vmn2r65 + neurons from three animals examined).

(D) RNA FISH showing that Vmn2r65 neurons are activated by the exposure of virgin males to the peak fraction of anion exchange chromatography.

(C) Activity profile of anion exchange chromatography fractions using Sepharose DEAE peak activity fractions as the input. The arrow marks the peak activity fraction (assayed in D), which was further analyzed by mass spectrometry.

(B) Activity profile of gel filtration chromatography fractions using female C57BL/6J salivary gland extract as a starting material. The ligand activity for Vmn2r65 was assayed by exposure of virgin males to selected fractions. Each dot represents the mean percentage of Vmn2r65 + Egr1 + neurons among all Vmn2r65 + neurons quantified on at least four VNO sections. Molecular weight for each fraction was estimated by SDS-PAGE. The ligand activity appears to elute broadly from this column, spanning 60–90 kDa.

(A) Purification strategy of compound(s) activating Vmn2r65-expressing neurons (see Method Details ). The mass spectrometry profile of the peak activity fractions obtained from adult female salivary gland extracts was compared to the most homologous fractions obtained from male gland extracts.

We produced full-length recombinant Smgc in E. coli and confirmed its biological activity on Vmn2r65-expressing neurons in virgin males, thus demonstrating Smgc as a ligand for Vmn2r65 ( Figures 4 E and 4F). Importantly, Smgc was also identified by MS of partially fractionated pup salivary glands ( Figures 4 G and S4 B), confirming that adult females and pups commonly produce Smgc as a pheromone.

The highly purified, active fraction from female salivary glands was strikingly enriched in submandibular gland protein C (Smgc), a large 70 kDa protein previously identified from rat and mouse salivary glands () ( Figures 4 C, 4D and S4 A). Interestingly, Smgc is expressed by juveniles of both sexes but exclusively by adult females (), consistent with the specificity of activation of Vmn2r65 by juvenile and adult tissue extracts shown earlier ( Figures 3 E, 3F, and S3 ).

Based on previously identified ligands of the large metabotropic glutamate receptor subfamily, we assumed that Vmn2r65 ligands would likely consist of polypeptides (). Moreover, the protein(s) of interest should be secreted in order to act as pheromone(s).

Vmn2r88 Is Activated by Specific Paralogs of Hemoglobin

Figure S5 Additional Data for the Identification of Hemoglobins as Ligands for Vmn2r88, Related to Figure 5 Show full caption (A) List of proteins identified by mass spectrometry in purified active fraction derived from C57BL/6J male salivary gland extracts. Hemoglobin Hbb-bt (in this diagram denoted as Hbbt1, per Uniprot nomenclature) emerges as a top candidate. (B) The peak activity fraction of Poros HQ chromatography is red. (C) Silver stained gel of the peak activity fraction derived from pup salivary gland extracts showed a unique 14 kDa band. (D) In situ hybridization of Vmn2r88 and Egr1 in the VNO of animals exposed to the peak fraction shown in (C). (E) Peptides corresponding to hemoglobins identified by mass spectrometry analysis of the purified pup fractions. This analysis confirmed that Hbb-bt (or Hbbt1) is a top candidate. (F) Gel filtration chromatography of recombinant hemoglobin Hbb-bt-Hba-a1. Purified recombinant hemoglobin appeared predominantly as a dimer rather than tetramer. Endogenous hemoglobin tetramer in C57BL6/J male blood extracts has a significantly shorter retention time than the major recombinant hemoglobin peak. Note that the Hba-a1 subunit in the recombinant hemoglobin is 11 amino acids longer than the native protein, resulting in a shorter retention time. (G) Quantitative analysis of the retention times of hemoglobins in gel filtration chromatography. The table shows the mean retention time of the tetramer and dimer peaks (shown as arrows in F) (N = 3 technical replicates). We next compared MS datasets obtained with active fractions of salivary gland extracts from C57BL/6J males and females with equivalent (but largely inactive) fractions obtained from 129S1/SvImJ mice. This strategy resulted in a list of secreted proteins that are candidate Vmn2r88 ligands ( Figure S5 A). We also noticed that the active purified fractions had a red tint ( Figure S5 B). Intriguingly, MS identified hemoglobins (Hbs) among the top candidate Vmn2r88 ligands ( Figure S5 A), and the Vmn2r88-stimulating activity was indeed present in blood lysate ( Figure 5 C). The purified active fraction from salivary gland extracts visualized on a SDS-PAGE gel showed prominent 14 kDa bands ( Figures 5 D and 5E), which matches the molecular weight of the monomeric Hb subunits alpha and beta.

Weaver et al., 1981 Weaver S.

Comer M.B.

Jahn C.L.

Hutchison 3rd, C.A.

Edgell M.H. The adult beta-globin genes of the “single” type mouse C57BL. Beta subunits of mouse Hb are strain specific (), and Hbb-bt (also known as Hbbt1; Figure S5 A), the C57BL/6J-specific Hb beta subunit identified by MS, satisfied the expected strain specificity of the Vmn2r88-ligand activity. To reconstitute Hb complexes occurring in vivo, we co-expressed recombinant alpha and beta subunits in E. coli and purified the resulting complexes ( Figure 5 F), the vast majority of which proved to be heterodimers of Hbb-bt and Hba-a1 with a minor contribution of tetramers ( Figures S5 F and S5G). We then presented the reconstituted Hb complexes to virgin male mice in order to test whether they displayed Vmn2r88-ligand activity.

Indeed, VNO neurons expressing Vmn2r88 showed robust activation after exposure of virgin males to the Hbb-bt-Hba-a1 complexes ( Figure 5 G). Interestingly, neurons expressing Vmn2r122/123 were also activated by Hbb-bt-Hbb-a1 ( Figure 5 G). Finally, Hbb-bt was also identified by MS in pup salivary gland extract fractions with Vmn2r88-ligand activity ( Figures S5 C–S5E), demonstrating that Hb represents the Vmn2r88-ligand activity in both adults and pups.

s and Hbbd. The Hbbs haplotype of the C57BL/6J strain is defined by the Hbb-bt gene, while the Hbbd haplotype of the 129S1/SvImJ strain is represented by the Hbb-b1 and Hbb-b2 genes ( Hempe et al., 2007 Hempe J.M.

Ory-Ascani J.

Hsia D. Genetic variation in mouse beta globin cysteine content modifies glutathione metabolism: implications for the use of mouse models. Raabe et al., 2011 Raabe B.M.

Artwohl J.E.

Purcell J.E.

Lovaglio J.

Fortman J.D. Effects of weekly blood collection in C57BL/6 mice. The Hb beta gene locus in mice exists in two major haplotypes: Hbband Hbb. The Hbbhaplotype of the C57BL/6J strain is defined by the Hbb-bt gene, while the Hbbhaplotype of the 129S1/SvImJ strain is represented by the Hbb-b1 and Hbb-b2 genes (). Because Hbb-bt and Hbb-b1 sequences differ in only three amino acids, we wondered whether Vmn2r88 can discriminate between these two ligands. We expressed Hbb-b1 and Hbb-b2, each complexed with Hba-a1 in E. coli, and exposed virgin male mice to these purified protein complexes at the same concentration as in our initial test with Hbb-bt/Hba-a1 (0.4 mg at 4 mg/mL). Notably, this concentration is over an order of magnitude below the reported concentration of Hb in the blood of adult mice (∼140 mg/mL) () and therefore likely falls within a physiological range as a VNO stimulus. Interestingly, in contrast to the robust activation by Hbb-bt-Hba-a1 ( Figure 5 G), we observed a much weaker activation of Vmn2r88-expressing neurons by Hbb-b1-Hba-a1 and no apparent activation by Hbb-b2-Hba-a1 ( Figure 5 H): Egr1 signals in Vmn2r88-expressing neurons in VNOs of animals exposed to Hbb-b2-Hba-a1 did not significantly exceed the background (1.77 ± 0.13 σ above background, N = 3∼4 per stimulus), and this activity was significantly weaker than the activities of Hbb-bt-Hba-a1 and Hbb-b1-Hba-a1 as Vmn2r88 ligands (p < 0.001; Figure 5 I). These results suggest that Hbs emitted by several mouse strains may act as VNO cues and that the strength of signaling through Vmn2r88-expressing neurons is strain dependent. In addition, the specificity of the Vmn2r88-stimulating activity seems to reside in the beta subunit of the Hb complex.

Gomes et al., 2010 Gomes I.

Dale C.S.

Casten K.

Geigner M.A.

Gozzo F.C.

Ferro E.S.

Heimann A.S.

Devi L.A. Hemoglobin-derived peptides as novel type of bioactive signaling molecules. The finding that Vmn2r88 is activated by Hb is highly unexpected, since Hb, although reported to be expressed in tissues other than mature and immature red blood cells (), is not known to be actively secreted outside the body in the absence of bleeding. Moreover, because attacks by males will naturally release Hb through pup wounding, activation of Vmn2r88 may result from, rather than be the cause of, male aggression. We have shown that Vmn2r88 is also robustly activated in virgin females exposed to pups, suggesting that Hb is naturally presented by pups even in the absence of male attacks ( Figure S2 B). These considerations prompted us to further examine the origin of this chemosignal.

Isogai et al., 2011 Isogai Y.

Si S.

Pont-Lezica L.

Tan T.

Kapoor V.

Murthy V.N.

Dulac C. Molecular organization of vomeronasal chemoreception. Blood from both male and female adult C57BL/6J mice strongly activated Vmn2r88-expressing neurons ( Figure 5 C). In addition, several lines of evidence suggest that Hb is constitutively deposited in mouse bedding in absence of overt bleeding. First, the Vmn2r88 ligand is enriched in the mixed bedding of adult male mice from five strains (). Second, we found that bedding from group-housed adult C57BL/6J males, but not from adult virgin C57BL/6J females, activates Vmn2r88-expressing neurons ( Figure 5 J). Third, strong Vmn2r88-ligand activity was found in bedding of co-housed pups and mothers, and in pups presented alone, while the ligand activity is not detectable in the bedding of C57BL/6J mothers after they were transferred to new cages with fresh bedding and without pups for 2 days ( Figure 5 J).

Strikingly, we consistently identified Hbs in the beddings of mothers co-housed with P1 pups by western blot, while Hbs were not detected in the bedding of similarly single-housed non-pregnant females and males ( Figure 5 L). The observation that Vmn2r88-stimulating activity is found in the bedding of mother with pups could indicate the presence of remnant blood following parturition. This hypothesis prompted us to test the stability of Hb over days. Indeed, we found that hemoglobin’s ability to activate Vmn2r88 remains stable for at least 3 weeks ( Figures 5 M and 5N), consistent with the notion of Hb signals able to persist from parturition until weaning.

Finally, in order to determine whether Hbs found in the bedding of co-housed pups and mothers originate from pups, mothers, or both, we tested bedding from cross-fostered pups. Since Hbb-b1, expressed by the 129S1/SvImJ strain, is less active in stimulating Vmn2r88 neurons than Hbb-bt, expressed by C57BL/6J mice, transfer of Hbs from C57BL/6J pups should be detectable in bedding of 129S1/SvImJ mother’s cages. However, cross-fostering C57BL/6J pups with 129S1/SvImJ mothers did not increase Vmn2r88-stimulating activity of bedding, while bedding of cross-fostered 129S1/SvImJ pups with C57BL/6J mothers did ( Figure 5 K). This indicates that mothers might be a more significant source of Hbs, likely deposited during parturition, than pups.

Hbs thus emerge as a new class of chemosignals that, since highly correlated with the birth and continuing presence of pups until weaning, can be exploited by males. Hbs, together with Smgc, are the first compounds isolated based on their participation in pup signaling to males. However, our attempts to swab recombinant Hbs and Smgc alone and together onto pup dummies did not result in any apparent increase in male aggression. This result indicated that the sole or combined activation of Vmn2r65-, Vmn2r88-, and Vmn2r122/123-expressing neurons from the seven receptor populations detecting pup cues is insufficient to fully represent the complex blend of pup signals leading to infanticide. Future studies may take advantage of the experimental system developed here to identify ligands corresponding to the other pup-activated VRs, i.e., V1rc1, V1rc30, and V1ri9.