The Relationship Between The Methylation Level Of The Promoter Region Of Vasopressin Type 1a Receptor (V1aR) Gene And Its MRNA Expression Is Related To The Spouse Relationship Of Brown Voles

The formation of enduring relationships between adult mates, such as pair bonding, is an indispensable aspect of human social behavior and has been involved in both physical and psychological health. Several lines of evidence show that vasopressin la receptors (V1aR) gene played a significant role in the regulation of monogamous social behaviors, including pair bonding. However, elegant brain receptor autoradiography studies suggest that the distribution patterns of the V1aR are remarkably different between monogamous and nonmonogamous voles. The molecular approach provides additional evidence implicating voles with the similar promoter structure of the V1aR gene display significant differences in mating system and social behavior. The exact contributions of the microsatellite to inter and intra-specific regional V1aR expression and pair bonding remain controversial. The hypothesis that interaction between V1aR gene and early environment may regulate monogamy social behavior is proposed by behavioral epigenetic research and our initial studies. Monogamous and nonmongamous voles have different V1aR densities in the lateral septum (LS) and ventral pallidum(VP). Nevertheless, VlaR in the ventral pallidum and lateral septum have been shown to be involved in the regulation of partner preference formation and paternal care. In particular, we proposed that increased V1aRs in the ventral pallidum could shift some individuals within a species to form pair-bonding and ultimately result in the stable selection of monogamous social organization. To test this hypothesis directly, we used viral vector-mediated gene transfer to over-express V1aR in the ventral pallidum in the paternal deprived (PD) male mandarin vole. In summary, here we proposed that early experiences such as paternal deprivation could alter the levels of V1aR gene methylation which subsequently affects levels of this gene’s expression, and as a result change the monogamous social behaviors.Our study mainly attempted to address following questions:whether different level of paternal investments affect VlaR gene expression; whether different paternal investments affect levels of V1aR gene methylation; whether different family structure patterns affect the distribution of V1aR receptor in the LS and VP? To explore the molecular biological mechanism underlying formation of pair-bonding, we will reveal the epigenetic mechanism underlying pair bonding via elucidating the association between different family structure and mRNA expression as well as DNA methylation of V1aR which is considered to be concerning to monogamous pair bonding. In order to add further evidences to this hypothesis, we test whether V1aR gene over-expression in the ventral forebrain via transferring the viral vector of this gene can substantially increase partner preference formation in the socially paternal deprivation male mandarin voles.Method:Thirty female and30male adults were randomly assigned to non-sibling breeding pairs. The30pairs Mandarin voles were randomly arranged into2groups:Biparental care group (PC) consisting of10pairs; and paternal deprivation group (PD) consisting20pairs. In the PC group all family members stayed together in their home cage; in the PD group the male parent was removed during the first24hours after the birth of the pups and the pups were reared by the mother alone. We used bisulfite sequence technique to analyze the different methylation island among promoter region of V1aR DNA gene following treatment with sodium bisulfite (NaHSO3) in mandarin voles from different groups.Our study also employed real-time quantitative PCR to examine the levels of the V1aR mRNA expression in the LS and VP of adult male mandarin vole with different social experience. The V1aR-vp group of PD male mandarin voles (n=8) received bilateral infusions into the ventral pallidum of the adeno-associated viral (AAV) vector containing the mandarin vole V1aR gene and under the control of a neuron-specific enolase promoter. The Ctrl-vp group (control; n=5) received bilateral infusions into the ventral forebrain of the control AAV vector expressing the lacZ gene under a cytomegalovirus promoter.Results:1. Sequence analysis of V1aR gene promoter region and exon1:Through analysis, we found that AVPR1a gene promoter region have3potential CpG islands with lengths of363bp,263bp and555bp. The three CpG islands contain25,25and48CpG sites respectively.2. Effects of early paternal environment on V1aR mRNA expression in the LS and VP in adult mandarin vole. The levels of VlaR mRNA expression in VP was significantly higher in PC than those in PD group, in contrast, The levels of V1aR mRNA expression in LS was remarkable lower in PC than those in PD group The results implicated that the different paternal investment significantly affected the type of V1aR receptor expression in different brain regions in voles. We revealed that the different levels of paternal investment impact V1aR mRNA expression in the brain regions associated with pair bonding.3. DNA methylation analysis of V1aR gene promoter region in the LS and VP:V1aR gene promoter region of5’non-coding region of CpG islands in PC mandarin vole exhibited hypomethylation. V1aR gene the exon1region of CpG islands displayed hypermethylation in specific brain region following PD treatment that is probably associated with modulation of pair bonding. Compared with the PC group, PD group showed lower level of methylation in the LS. Parental investment affected CpG island methylation in the LS. PD group showed higher levels of methylation in the first paragraph of the first exon CpG islands in V1aR gene in the VP. Variation in parental investment may affect the degree of methylation of CpG islands in mandarin vole offspring.4. In the LS and VP, mRNA expression of V1aR gene was associated with DNA methylation states of V1aR gene5’non-coding region. However, the differences in CpG methylation of the V1aR gene were not specific to any individual CpG sites. Rather, LS showed increased cytosine methylation in DNA from PC compared to PD group; on the contrary, VP exhibited decreased DNA methylation from PC vs PD group.5. Effects of the over-expression of V1aR-VP on pair bonding and social behavior. The SIT showed that PD male mandarin voles treated with over-expressed V1aR bilaterally in the ventral pallidum (V1aR-VP) showed significantly more body contact, investigation, attack behavior and self-grooming. The PPT showed that VlaR-VP male group displayed the formation of a preference for the female partner. Adult male offspring from VlaR-VP group spent significantly more time with the partner than with the stranger and showed more aggressive behavior to the stranger than the partner, indicating typical performance of PC-reared male voles.Conclusion:In mandarin voles, the V1aR gene promoter region include two CpG islands, which including25CpG sites respectively and were found to display the different level of DNA methylation. However, the differences in CpG methylation of the VlaR gene were not specific to any individual CpG sites. Compared to PD group, Hypomethylation of CpG islands in V1aR gene5’non-coding region was found in the VP in the PC group; but hypermethylation of CpG islands in the exon1region of V1aR gene was found in LS following PC treatment. Thus, the degree of DNA methylation of V1aR gene is likely associated with early social environments.As known to all, the negative correlation exists between DNA methylation and gene expression. Under different early social conditions, the levels of V1aR mRNA expression in the VP were significantly higher in PC than those in PD group; in contrast, the levels of V1aR mRNA expression in the LS were remarkable lower in PC than those in PD group. These effects are likely not only due to the methylation states of vole V1aR gene5’non-coding region, but also by the degree of DNA methylation of exon1region. We suggested that different early life environment had critical influences on the relative receptor densities via epigenetic mechanism in brain region associated with mating system in mandarin voles. We show that a change in the expression of a single gene in the larger context of pre-existing genetic and neural circuits can profoundly alter social behavior, providing a potential molecular mechanism for the development of complex social behavior.Present laboratory research results show that the male voles received paternal deprivation significant differ from PC-reared male vole according to the SIT and PPT. We used viral vector-mediated gene transfer to over-express V1aR in the ventral pallidum in the PD mandarin vole. The SIT showed that V1aR-VP treatment significantly increased body contact, investigation, attack behavior and self-grooming. The PPT showed that V1aR-VP treatment improved the formation of a preference for the female partner in males. Adult male offspring from V1aR-VP group spent significantly more time with the partner than with the stranger and showed more aggressive behavior to the stranger than the partner displaying typical performance of PC-reared male voles. Thus, we conclude that early social experiences such as paternal deprivation may affect the levels of methylation of VlaR gene in specific brain regions, and subsequently alter expression levels of the gene, and as a result change the monogamy related social behaviors. To some extent, the present findings elucidate the episenetic mechanism of monogamous social behavior.