Maternally inherited peptides as strain-specific chemosignals

Hideto Kabaa,1,2, Hiroko Fujitaa,1, Takeshi Agatsumab, and Hiroaki Matsunamic,d,e,2

aDepartment of Physiology, Kochi Medical School, Kochi University, Nankoku, 783-8505 Kochi, Japan; bDepartment of Environmental Health Sciences, Kochi Medical School, Kochi University, Nankoku, 783-8505 Kochi, Japan; cDepartment of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710; dDepartment of Neurobiology, Duke University Medical Center, Durham, NC 27710; and eDuke Institute for Brain Sciences, Duke University, Durham, NC 27710

Edited by Frank Zufall, Saarland University, Homburg, Germany, and accepted by Editorial Board Member John R. Carlson October 13, 2020 (received for review July 13, 2020) Most mammals rely on chemosensory cues for individual recogni- of the ESP1 receptor, V2Rp5, which is expressed in vomeronasal tion, which is essential to many aspects of social behavior, such as sensory neurons (VSNs). maternal bonding, mate recognition, and inbreeding avoidance. We have focused on mitochondrially encoded peptides as a Both volatile molecules and nonvolatile peptides secreted by candidate source of individual chemosignals, based on highly individual conspecifics are detected by olfactory sensory neurons polymorphic genes. Because NADH dehydrogenase 1 (ND1) in the olfactory epithelium and the . The perti- and NADH dehydrogenase 2 (ND2) are encoded in the mito- nent cues used for individual recognition remain largely unidenti- chondrial genome, they are maternally inherited, formylated at fied. Here we show that nonformylated, but not N-formylated, the N-terminal methionine at the time of synthesis, and poly- mitochondrially encoded peptides—that is, the nine N-terminal morphic among inbred strains (Fig. 1). The ND1 peptide is de- amino acids of NADH dehydrogenases 1 and 2—canbeusedto rived from the nine N-terminal amino acids of the ND1 protein: convey strain-specific information among individual mice. We dem- MFFINXLTL. The identity of the sixth amino acid varies among onstrate that these nonformylated peptides are sufficient to induce different strains and can be either A, I, T, or V. ND2 has similar a strain-selective block. We also observed that the preg- amino acid substitutions. These features raise the possibility that nancy block by an unfamiliar peptide derived from a male of a dif- mitochondrially encoded peptides can be used to convey mouse ferent strain was prevented by a formed at the time of strain-specific information among individuals. NEUROSCIENCE mating with that male. Our findings also demonstrate that This possibility can be explored in the context of the selective pregnancy-blocking chemosignals in the are maternally pregnancy block effect, which is also known as the Bruce effect inherited, as evidenced by the production of reciprocal sons from (21) and depends on a functional vomeronasal system (22–25). two inbred strains and our test of their urine’s ability to block preg- In this behavioral paradigm, a high incidence of pregnancy fail- nancy. We propose that this link between polymorphic mitochon- ure occurs when recently mated mice are exposed to drial peptides and individual recognition provides the molecular means to communicate an individual’s maternal lineage and strain. chemosignals from a second unfamiliar male. The female re- ceives the signal from an unfamiliar male through her VNO, mitochondria | olfaction | maternal inheritance | | Bruce effect resulting in a suppression of the and

Significance he ability to distinguish and recognize individuals is critical Tfor animals’ adaptive regulation of social behavior. Although individual information may be coded in a number of ways, che- For most mammals, olfaction plays an important role in indi- mosensory cues are a primary means by which many animals vidual recognition. The social chemosignals secreted by indi- evaluate others’ identity, relatedness, sex, and social status (1–6). vidual conspecifics are complex variable mixtures that are still Each individual animal has a great complex of chemosensory poorly understood. Here we identify nonformylated, but not N-formylated, mitochondrially encoded peptides: That is, the cues, some self-generated and others acquired from the envi- nine N-terminal amino acids of NADH dehydrogenases 1 and 2 ronment and conspecifics (7). The resulting chemical signatures as reliable chemosignals for individual recognition by using a are complex variable mixtures that are still poorly understood. vomeronasal organ-mediated pregnancy block, known as the Three highly polymorphic multigene families in mice are Bruce effect. Our findings also revealed that pregnancy- known to contribute to individual differences in chemosensory blocking chemosignals in the urine are maternally inherited. cues that are detected through specific vomeronasal receptors We propose that this individuality cue provides the molecular expressed in sensory neurons of the vomeronasal organ (VNO). means to communicate an individual’s maternal lineage The major histocompatibility complex (MHC) genotype has and strain. been linked to mate recognition in the pregnancy block effect, as congenic mice differing from the mating male only in their MHC Author contributions: H.K. and H.M. designed research; H.K. and H.F. performed research; genotype were effective in blocking pregnancy (8). Further evi- H.K., H.F., T.A., and H.M. contributed new reagents/analytic tools; H.K. and H.F. analyzed dence has shown that the MHC peptide ligands themselves data; and H.K., H.F., T.A., and H.M. wrote the paper. Competing interest statement: H.M. receives royalties from Chemcom. Other authors convey strain-specific information in the context of the preg- declare no competing interest. nancy block effect (9, 10). Major urinary proteins provide an This article is a PNAS Direct Submission. F.Z. is a guest editor invited by the individual genetic identity signature that underlies individual Editorial Board. – recognition, territorial behavior, and (11 16). Published under the PNAS license. A series of studies demonstrated that exocrine-gland secreting 1H.K. and H.F. contributed equally to this work. peptide 1 (ESP1) that is released into the tear fluids of adult 2 To whom correspondence may be addressed. Email: [email protected] or hiroaki. male mice is one of the key factors that cause pregnancy block [email protected]. – (17 20). Female mice exhibit high pregnancy failure rates upon This article contains supporting information online at https://www.pnas.org/lookup/suppl/ encountering males that secrete different levels of ESP1 com- doi:10.1073/pnas.2014712117/-/DCSupplemental. pared to the mated male. This effect is mediated via the activation

www.pnas.org/cgi/doi/10.1073/pnas.2014712117 PNAS Latest Articles | 1of6 Downloaded by guest on October 2, 2021 male urine at a final concentration of 50 μM and testing its pregnancy-blocking effectiveness following mating with a BALB/ c male. The N-formylated variants of the ND1 and ND2 peptides and the prototypical bacterial N-formyl-methionyl-leucyl-phe- nylalanine (MLF) were ineffective at inducing pregnancy failure (Fig. 3A). N-formylated ND1-6A was still ineffective even when applied at a high dose (150 μM). The low rate of pregnancy failure in the group exposed to the N-formylated ND1 or ND2 variant is not ascribed to an overall decrease in the susceptibility to pregnancy block, because pregnancy-blocking effectiveness Fig. 1. N-terminal polymorphisms in the mouse mitochondrial ND1 and was confirmed by exposure to CBA male urine that served as a ND2 proteins. The polymorphic positions in ND1 and ND2 are shown in bold. positive control. Parentheses indicate H-2 haplotypes, where known. f-M, formyl-methionine. In contrast, the nonformylated forms of ND1-6A and ND2-7T derived from the NZB strain significantly increased the effec- tiveness of BALB/c male urine in blocking pregnancy, whereas levels that are necessary for embryonic implantation. Hence, the the nonformylated form of ND1-6I or ND2-7A derived from the presence of an unfamiliar male can terminate by a BALB/c strain did not (Fig. 3B). In addition, the pregnancy block male of a different strain, but it does not block pregnancies that effect of nonformylated ND1-6A was dose-dependent; a low he himself (or a mouse of the identical strain) has initiated. This μ B “ ” is because the female learns to recognize and remember her dose (5 M) was without effect (Fig. 3 ). Thus, familiar urine “ ” mate’s chemosignals during a sensitive period at the time of could be converted to unfamiliar urine by the addition of a mating and gates their effects in order to maintain the pregnancy. single nonformylated peptide derived from a strain other than BALB/c. The potency of nonformylated ND1-6A in blocking Results pregnancy was comparable to that of AAPDNRETF (9), an b N-Terminal Polymorphisms in the ND1 and ND2 Proteins. The mito- MHC class I peptide of the disparate H-2 haplotype (Fig. 3B). A chondrial DNA sequencing confirmed that the inbred strains of scrambled version of nonformylated ND1-6A MFFINALTL, mice (i.e., BALB/cCrSlc, CBA/NSlc, and NZB/NSlc) from Japan namely LMATLFNIF, failed to induce pregnancy failure, suggesting SLC have the same N-terminal amino acid sequences deduced from the nucleotide sequences of the mitochondrial nd1 and nd2 genes as the corresponding inbred strains from The Jackson Laboratory: BALB/cJ (GenBank accession no. AJ512208.1) (26), A CBA/J (GenBank accession no. AY466499.1) (27), and NZB/ Stimulus exposure: Mating exposure: BALB/c B1NJ (GenBank accession no. L07095.1) (28) (Fig. 1 and SI Appendix, Fig. S1). MilP (GenBank accession no. L07096.1) (28) and BALB.SHC (28) mice encode different allelic forms of ND1 No exposure (10) (i.e., ND1-6V and ND1-6T, respectively). * BALB/c male (10) * Both Male and Female Mice of the NZB Strain Induced Pregnancy * Block in H-2–Compatible BALB/c . We first tested the preg- NZB male (11) nancy block effect on BALB/c mice by NZB mice, which differ from H-2d-compatible BALB/c mice by a single amino acid substitution in the ND1 and ND2 peptides (Fig. 1). Female 020406080100 BALB/c mice were mated with BALB/c males and then exposed Pregnancy failure (%) to the familiar BALB/c males or unfamiliar NZB males. The exposure to NZB males resulted in a significantly higher level of B pregnancy failure compared to no exposure and to exposure to the familiar BALB/c males (Fig. 2A). This effect was reproduced Stimulus exposure: Mating exposure: BALB/c by the application of urine taken from not only males but also females of the NZB strain on the oronasal groove of the females BALB/c male urine (Fig. 2B). These results suggest that pregnancy-blocking che- (9) d * mosignals, independent of the H-2 haplotype and , are * * present in urine. NZB male urine (16) * The Variants of Mitochondrial Peptides Differentially Induced NZB female urine (17) Pregnancy Block. The release of N-formylated peptides from the mitochondria of damaged or dying cells plays an important role in tissue homeostasis (29). Mitochondrially encoded peptides are 020406080100 presented to cytotoxic T lymphocytes by an MHC class Ib mol- Pregnancy failure (%) ecule, H2-M3 (28, 30, 31). The amino-terminal N-formyl-me- thionine is essential for binding to H2-M3 (32). However, more Fig. 2. Pregnancy block caused by the H-2d–compatible strain of mice and recent studies showed that mammalian peptide deformylases are their urine. (A) Pregnancy block occurs when female mice of the BALB/c strain (H-2d haplotype) are mated with BALB/c males and then exposed to H- capable of removing the N-terminal formyl group of mito- d chondrially encoded peptides (33, 34). The question thus arises: 2 -compatible NZB males that differ in the maternally transmitted antigen. (B) NZB male-induced pregnancy block is reproduced by the application of Are mitochondrially encoded peptides the chemosignal that urine taken from not only males but also females of the NZB strain on the enables NZB-induced pregnancy block in BALB/c females? oronasal groove of mated females. The numbers in parentheses indicate the We addressed this question by adding the N-formylated or numbers of animals tested. *P < 0.05, **P < 0.01, Fisher’s exact probability nonformylated variant of the ND1 or ND2 peptide to BALB/c test with Bonferroni correction for multiple comparisons.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.2014712117 Kaba et al. Downloaded by guest on October 2, 2021 AB Stimulus exposure: Mating exposure: BALB/c Stimulus exposure: Mating exposure: BALB/c

BALB/c male urine (15) BALB/c male urine (27)

N-formyl ND1-6I (12) Non-formyl ND1-6I (16)

N-formyl ND1-6A (11) Non-formyl ND1-6A (36) * * N -formyl ND1-6A (12) Non-formyl ND1-6A (12) (high dose) (low dose) N-formyl ND1-6V (10) Non-formyl ND1-6A (10) scrambled N-formyl ND1-6T (15) Non-formyl ND1-6V (21)

N-formyl ND2-7A (10) Non-formyl ND1-6T (19)

N-formyl ND2-7T (9) Non-formyl ND2-7A (11)

N -formyl MLF (10) Non-formyl ND2-7T (13) * CBA male urine (10) AAPDNRETF (15) * * *

0 20 40 60 80 0 20 40 60 80 Pregnancy failure (%) Pregnancy failure (%)

Fig. 3. Nonformylated (B), but not N-formylated (A), variants of mitochondrially encoded peptides differentially induce pregnancy block. The numbers in parentheses indicate the numbers of animals tested. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the BALB/c male urine control; Fisher’s exact probability test with Bonferroni correction for multiple comparisons.

that the effect of nonformylated ND1-6A depends on the integrity at blocking pregnancy, and the results also suggest that females NEUROSCIENCE of the peptide sequence (Fig. 3B). habituate to their own expression of pregnancy-blocking che- Taken together, these results show that mitochondrially mosignals including mitochondrially encoded peptides. The ef- encoded peptides can convey strain-specific information in the fectiveness of CBA male (disparate H-2k haplotype) urine in context of the pregnancy block effect, and that peptides differing inducing a pregnancy block in BALB/c-mated BALB/c females by a single amino acid residue can be distinguished. (Fig. 3A) or NZB-mated BALB/c females (Fig. 4) points to the involvement of factors other than the ND1 and ND2 peptides Female Mice Form a Memory of Mitochondrial Peptides of the Mating that are identical between the CBA and BALB/c strains. Male. We then investigated whether the effectiveness of non- formylated ND1-6A in blocking pregnancy is altered following Maternal Inheritance of the Pregnancy Block Effect. If the pregnancy- mating with an NZB male. As expected, the addition of non- blocking chemosignals in NZB male urine are maternally inheri- formylated ND1-6A derived from the NZB strain to NZB male ted, then sons of reciprocal crosses between BALB/c and NZB urine did not increase the incidence of pregnancy failure in mice should only block pregnancy when their mother, but not NZB-mated BALB/c females (Fig. 4). These results suggest that their father, is of the NZB strain. We tested this possibility. This BALB/c females formed a memory of nonformylated ND1-6A type of inheritance was validated, as the exposure of BALB/c-mated that was naturally released from NZB males at the time of mating, thereby preventing pregnancy failure upon subsequent exposure to this peptide. Stimulus exposure: Mating exposure: NZB It has been demonstrated that pregnancy block occurred after the application of BALB/c male urine or prototypical represen- d tative ligands for the H-2 haplotype of BALB/c mice, but not NZB male urine (11) after the application of C57BL/6 male urine or ligands for the H- 2b haplotype of C57BL/6 mice when BALB/c females were mated with C57BL/6 males (9). This was also true for BALB/c Non-formyl ND1-6A (19) females that had mated with CBA males (SI Appendix,Fig.S2). d These findings indicated that BALB/c male urine or H-2 –type BALB/c male urine (10) ligands are capable of inducing pregnancy failure when BALB/c females were mated with disparate H-2 haplotype males, whereas the female haplotype is irrelevant in this behavioral response. CBA male urine (16) * Accordingly, this behavioral response is attributable, at least in part, to pregnancy-blocking chemosignals that exhibit male- specific expression. 020406080 In contrast, we observed that the application of BALB/c male Pregnancy failure (%) urine did not increase the incidence of pregnancy failure in NZB-mated BALB/c females, while the application of CBA male Fig. 4. BALB/c females learn to recognize and remember nonformylated urine resulted in a significantly higher level of pregnancy failure ND1-6A that is naturally released from an NZB male at the time of mating. Notably, exposure to male BALB/c urine does not increase the incidence of compared to the application of NZB male urine (Fig. 4). These pregnancy failure in NZB-mated BALB/c females. The numbers in parenthe- results indicate that the female haplotype influences the effec- ses indicate the numbers of animals tested. *P < 0.05 vs. the NZB male urine tiveness of male urine in inducing a pregnancy block, an effect control; Fisher’s exact probability test with Bonferroni correction for that is in accord with the fact that female urine itself is effective multiple comparisons.

Kaba et al. PNAS Latest Articles | 3of6 Downloaded by guest on October 2, 2021 BALB/c females to (NZB\ × BALB/c_)F1 males resulted in a sig- AAA+ proteases are exported from mitochondria by the mi- nificantly higher level of pregnancy failure than exposure to (BALB/ tochondrial ABC (ATP-binding cassette) transporters (38, 39). N c\ × NZB_)F1 males (Fig. 5A). This effect was reproduced by urine The results of the present investigation show that the -formylated taken from the F1 males (Fig. 5B). The results confirmed that forms of mitochondrial peptides are ineffective in inducing pregnancy maternally inherited components of urinary cues are one of the block. It had been surmised that peptide deformylase does not exist in cues responsible for inducing a pregnancy block. eukaryotes, but subsequent studies obtained evidence that lower and higher eukaryotes, as well as bacteria, share similar N-terminal pro- Discussion tein processing machinery (40, 41). In fact, mammalian mitochondrial Our present findings revealed that mitochondrially encoded peptide deformylase is active, and its inhibition impairs mitochondrial peptides can function as strain-specific chemosignals that are translation and the assembly of oxidative phosphorylation complexes N maternally inherited and independent of androgen. The unfa- (33, 34, 41, 42) or causes apoptosis (43). The -formylated forms of miliar (nonself) variants of nonformylated peptides differentially mitochondrial peptides, such as ND1-6I and ND1-6T, stimulate induce a pregnancy block, while the familiar (self) variants are a subset of sensory neurons in the VNO (44), but they had no applicable effects on pregnancy block in the present study. These ineffective. The addition of a single nonformylated peptide from N an unfamiliar male in the urine of a familiar male is sufficient to findings suggest that there is no link between -formylated mi- alert the mating female that a strange male is present. Exposure tochondrial peptide-induced responses in the VNO and the to unfamiliar peptides at mating with a male of a different pregnancy block effect. Unlike the nonformylated mitochondrial peptides eliciting the neuroendocrine change, a distinct subset of strain causes the peptides to be recognized as familiar peptides N-formylated bacterial peptides activate formyl peptide recep- by the female. These findings suggest that the mitochondrial tors expressed in a subset of cells in the VNO (45, 46) and drive peptides are naturally released from a mouse and can form part of avoidance behavior in mice (46). the suite of cues for the recognition of strain differences. Such Herein, H-2d–compatible NZB male-induced pregnancy block mitochondrial peptides that can signal individual information may in BALB/c females was reproduced by the urine of NZB males, be of importance in other vertebrate species and behavioral which differ from BALB/c mice by a single amino acid substi- contexts. tution in the ND1 and ND2 peptides. However, the mitochon- Regarding the ability of mitochondria to direct self-elimination, drial peptides in mouse urine remain to be identified. It is also mitochondrially encoded proteins at the inner mitochondrial not yet known whether these peptides are the sole determinant membrane can be removed in two ways to maintain mito- of NZB-induced pregnancy block in BALB/c females. Notably, chondrial protein homeostasis: Through the intramitochondrial however, the NZB-induced pregnancy block could be ascribed to proteolytic pathway involving the mitochondrial AAA+ pro- polymorphisms in the mitochondrial genome, as judged by its teases (ATPases associated with a variety of cellular activities) maternal inheritance. When considering the biological signifi- (35), or through the autophagy-lysosomal pathway (36). A cance of maternally inherited peptides in olfactory communica- feature of the mitochondrial AAA+ proteases is the contrast- tion, it is of interest that the attraction of mice to urinary odors ing fates of different substrates. Proteins may be completely of other mice is subject to a parent-of-origin effect, which causes degraded to small peptide fragments, undergo partial process- both males and females to prefer the odor of urine from mice of ing to a fixed point in the structure, or be dislocated from the an unrelated strain to that of urine from mice of the same strain membrane without proteolysis (37). Peptides generated by the as their mother, thereby favoring outbreeding and heterozy- gosity (47). Recent experimental evidence from a seminatural population study suggests that the Bruce effect may be an A adaptive breeding strategy for females (48). Taken to- Stimulus exposure: Mating exposure: BALB/c gether, the past and present findings indicate that the maternal inheritance of mitochondrial DNA polymorphisms that directly BALB/c male (16) influence the olfactory block to pregnancy may promote not only individual recognition but also mechanisms of inbreeding avoidance. (BALB/c NZB )F1 male (15) * * We observed that urine taken from not only males but also (NZB BALB/c )F1 male (17) females of the NZB strain induced the pregnancy block effect in BALB/c-mated BALB/c females, suggesting the involvement 020406080100 of androgen-independent urinary cues in blocking pregnan- Pregnancy failure (%) cies. The extent to which pregnancy block depends on androgen- B dependent cues in male mouse urine is unclear (49). Urine from Stimulus exposure: Mating exposure: BALB/c sexually mature males has pregnancy-blocking activity. This activity is lost following castration (50, 51) and restored fol- BALB/c male urine (12) lowing testosterone injection (52). Cues from sexually imma- ture males (50) and from unfamiliar females of a different (BALB/c NZB )F1 male urine (17) * strain (21) have also been shown to be ineffective. However, * urine from congenic females of a different MHC type can in- (NZB BALB/c )F1 male urine (16) duce pregnancy block (8), and the direct application of cas- trated or juvenile male urine on the oronasal groove induces 020406080100 pregnancy block (10). Pregnancy failure (%) One of our important present findings is that the non- formylated forms of strain-specific ND1 and ND2 variants that Fig. 5. Pregnancy-blocking chemosignals in male NZB urine are maternally differ from those of the stud’s strain by a single amino acid inherited. (A) Reciprocal F1 males between BALB/c and NZB mice block pregnancy only when their mother, not father, is of the NZB strain. (B)F substitution can elicit pregnancy block. This raises the question 1 of why the nonformylated ND1 variants (ND1-6V and ND1-6T) male-induced pregnancy block is reproduced by urine taken from the F1 males. The numbers in parentheses indicate the numbers of animals tested. from MilP and BALB.SHC strains, respectively, did not signifi- *P < 0.05, **P < 0.01, Fisher’s exact probability test with Bonferroni cor- cantly increase the rate of pregnancy failure in BALB/c females rection for multiple comparisons. mated with BALB/c males, in contrast to the variant from the

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.2014712117 Kaba et al. Downloaded by guest on October 2, 2021 NZB strain (i.e., ND1-6A). Nevertheless, the pregnancy-blocking Materials and Methods effectiveness of the nonformylated ND1 variant from the MilP All procedures were performed with the approval (no. H-00058) of the Kochi strain was close to the threshold for statistical significance Medical School Animal Care and Use Committee and conformed to inter- (Fig. 3B)(P = 0.0558 vs. the BALB/c urine control; Fisher’s exact national guidelines on the ethical use of animals. probability test with Bonferroni correction for multiple com- parisons). The relationship among three individuals (mated fe- Mitochondrial DNA Sequencing. This sequencing study used three strains of male, mating male, and unfamiliar male) has a pronounced mice: BALB/cCrSlc (three females, 6- to 15-wk old), CBA/NSlc (three males, influence on the rate of pregnancy failure (53). Under such 47-wk old), and NZB/NSlc (four males, 15- to 20-wk old). Genomic DNA was circumstances, differences in the rate of pregnancy failure can be extracted individually from the liver of each mouse with an Easy-DNA Kit ascribed to differences in responsiveness of VSNs to the distinct (Invitrogen). The mitochondrial DNA was amplified using primers newly ND1 and ND2 variants. designed in this study for two gene regions, nd1 and nd2, on the basis of a With respect to peptide sensing in VSNs, V2rf2-expressing complete mitochondrial DNA sequence of BALB/cJ (GenBank accession no. VSNs, which coexpress MHC class Ib genes termed H2-Mv AJ512208.1) (26). genes, displayed a decreased sensitivity to the N-formylated The forward and reverse primers for nd1 (587 bp) were f2502ND1 (CGT GATCTGAGTTCAGACCG) and r3088ND1 (ACTGATAGGCTAGATGTTGC); those form of ND1-6T in H2-Mv gene knockout mice, indicating that N for nd2 (578 bp) were f3572ND2 (AGCATCTTATCCACGCTTCC) and r4149ND2 these VSNs detect -formylated ND1-6T via H2-Mvs (54). In (TTATAGTTGAGTACGATGGC), respectively. A PCR was performed using contrast, nonformylated mitochondrial peptides bind to H2-M3, ExTaq DNA polymerase (Takara) under the following conditions: 94 °C for 2 an MHC class Ib molecule, but they have to be added in 10 -to 3 min, followed by 31 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 4 10 -fold higher concentrations to cause the same level of cyto- 1 min, then 72 °C for 5 min. The obtained PCR products were electro- toxic T lymphocyte responses to N-formylated mitochondrial phoresed and purified from agarose gel with thermostable β-agarase peptides (55, 56). Thus, it is unlikely that the nonformylated (Nippon Gene). forms of strain-specific ND1 and ND2 variants induce pregnancy The purified PCR products were subjected to direct-sequencing with an block via H2-Mvs. V2r1b-expressing VSNs, which do not coex- ABI PRISM 3100-Avant DNA sequencer using a Big Dye Terminator v3.1 Cycle press H2-Mv genes, were differentially activated by peptides that Sequencing Kit (Applied Biosystems). The sequencing primers were the same are ligands of distinct MHC molecules (57). These findings are as those used for the PCR in the two regions. The reference sequence used in consistent with the idea that differential rates of pregnancy this study was obtained from the BALB/cJ mitochondrial genome (GenBank failure can be induced in response to distinct MHC peptide li- accession no. AJ512208.1) (26). gands. However, no study to date has demonstrated such dif-

Pregnancy Block. The inbred mouse strains BALB/cCrSlc, NZB/NSlc, and CBA/ NEUROSCIENCE ferential responses of VSNs to the strain-specific mitochondrial ± peptides, and much more work needs to be done to determine NSlc were purchased from Japan SLC. The animals were housed singly at 21 1 °C with a reversed 12:12 light cycle (lights on at 2100 hours, off at how VSNs and their receptors discriminate mitochondrial pep- 0900 hours). The estrous cycles of the virgin females of the BALB/c strain tide sequences and which ligand features are critical for sensory were monitored daily by taking vaginal smears between 0800 and neuron activation. 0900 hours. Sexually receptive females were identified by a typical pro- MHC peptide ligands, ESP1, and strain-specific mitochondrial estrous/estrous vaginal smear with approximately half cornified epithelial peptides are each capable of conveying individuality information and half nucleated epithelial cells. Mating was carried out naturally by in the pregnancy block effect, but these peptides act as a mixture placing a single sexually receptive female into the home cage of a male at under natural conditions. How do these peptides differentially 1400 hours and checking her for vaginal plugs at 2100 hours when the contribute to strain recognition? Single-unit recordings from the mating male was removed. Mated females were left in the cages of the accessory , the critical site for conspecific chemo- mating males until 0800 hours on the day following mating before removal sensory communication, of behaving, conscious mice revealed to a clean cage. that many mitral/tufted neurons were excited or inhibited by At 1500 hours on the day following mating, either the familiar stud or a specific combinations of sex and inbred strain identity of anes- strange male was introduced to the female’s cage and left with her for 48 h. thetized stimulus mice (3). Although it is difficult to see how Exposure to urine or supplemented urine was achieved by depositing 30 μL these peptides differentially contribute to strain identity, it ap- of liquid on the oronasal groove while holding the female by the nape of pears that information about individual identity is integrated in the neck. Mated females were exposed at 0800, 1400, 2000, and 2400 hours the accessory olfactory bulb based on the distinct features of on each of the 2 d following mating. Seven days after mating, the females were killed and their uteri were examined for implantation status. Whole these peptides (H-2 haplotypes, different levels of ESP1, and urine was collected from adult males and females, pooled by strain, and variant types of the ND1 and ND2 peptides), promoting indi- stored at −80 °C. Synthetic peptides purchased from GenScript and Operon vidual discrimination and recognition. Biotechnologies, stored at 1, 10, or 30 mM in DMSO, and further diluted in PBS. Urine samples were mixed 1:1 (vol/vol) with peptide solutions (final Conclusions concentrations: 5 [low dose], 50- or 150- [high dose] μM peptides and 0.5% The results reported here lead to three major conclusions. First, DMSO) (SI Appendix, Fig. S3). unfamiliar (nonself) nonformylated mitochondrial peptides dif- fering by a single amino acid residue from a familiar (self) Statistical Analyses. Data were analyzed with Fischer’s exact probability test peptide function as strain-specific chemosignals and thus dif- with Bonferroni correction for multiple comparisons. Significance was ac- ferentially induce a strain-selective pregnancy block. Second, an cepted at P < 0.05. unfamiliar peptide is converted to a familiar peptide by a memory formed at the time of mating with a male of a different Data Availability. All study data are included in the article and SI Appendix. strain expressing this peptide. Third, maternally inherited pep- tide components of urinary cues are capable of inducing a ACKNOWLEDGMENTS. We thank Dr. Peter Brennan for his comments on an earlier draft of this manuscript. This research was supported in part by a pregnancy block, providing convincing evidence of a direct link Grant-in-Aid for Challenging Exploratory Research from Japan Society for between polymorphic mitochondrial peptides and individual the Promotion of Science Grant 17K19635 (to H.K.) and by NIH Grants recognition. DC014423 and DC016224 (to H.M.) and NSF Grant 1556207 (to H.M.).

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