Report on the Study of the Impact of ANCV1R Gene Deficiency on Pheromone Detection and Sexual Behavior in Mice

Background Introduction

In mammals, pheromones are crucial chemical signals that regulate social and sexual behaviors. Pheromone detection is mediated by the vomeronasal organ (VNO), which uses vomeronasal sensory neurons (VSNs) to recognize chemical cues via specific vomeronasal receptors (VRs). These signals are relayed from the accessory olfactory bulb (AOB) to the amygdala and hypothalamus, triggering instinctive behaviors. Although Ancient Vomeronasal Receptor Type-1 (ANCV1R) has been evolutionarily conserved in vertebrates, its exact function remains unknown. Previous studies have shown that pseudogenization of the ANCV1R gene correlates with VNO degeneration. The goal of this study was to explore how ANCV1R deficiency impacts female mouse sexual behavior and pheromone sensitivity through behavioral, neurophysiological, and molecular analyses.

Source of the Study

This research was conducted by Hiro Kondo, Tetsuo Iwata, Koji Sato, and colleagues with affiliations to institutions such as the Institute of Science Tokyo (Japan), the University of Tokyo (Japan), and RWTH Aachen University (Germany). The study was published in the journal Current Biology on January 6, 2025 (DOI: 10.1016/j.cub.2024.10.077) and is available as an open-access publication.

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Overview of the Research Process

1. Subjects and Gene Knockout Model

The team generated ANCV1R-deficient mice (ANCV1R−/−) using the CRISPR-Cas9 system. Immunohistochemical analysis confirmed the complete absence of ANCV1R protein in these mice. In situ hybridization (ISH) verified that ANCV1R deficiency did not significantly impact the differentiation or survival of VSNs.

2. Behavioral Analyses

(1) Sexual Behavior

The sexual behavior of adult female mice was analyzed during interactions with male mice. Normally, male mice display sniffing and mounting behavior in the presence of receptive females, and females typically accept these behaviors. However, ANCV1R−/− females exhibited amplified sexual rejection, performing behaviors such as evasive running, upright stance to resist, and rejection of mounting attempts.

(2) Pheromone Preference

In a two-choice preference test, female mice were exposed to male urine and female urine simultaneously. Normal female mice showed a preference for male urine, but ANCV1R−/− females did not exhibit any preference, suggesting a crucial role for ANCV1R in pheromone detection.

(3) Maternal Behavior

Lactating ANCV1R−/− female mice exhibited deficiency in maternal aggression, showing significantly reduced attacks on intruders compared to wild-type females.

3. Physiological and Neural Signal Transmission Analyses

(1) Electrophysiological Responses in the VNO

Electro-vomeronasogram (EVG) recordings revealed significantly reduced responses in ANCV1R−/− females to male urine and specific pheromones such as β-estradiol-3-sulfate. Fluorescent staining and phosphorylated ribosomal protein S6 (pS6) analysis further confirmed that ANCV1R deficiency significantly decreased the sensitivity of VSNs to chemical ligands.

(2) Central Neural Activity

Using c-fos immediate early gene ISH, the researchers assessed neural activity in brain regions related to pheromone perception. Upon exposure to male urine, ANCV1R−/− females exhibited reduced neural activation in the AOB’s granule cell layer and the medial amygdala (MeA). Moreover, interactions with male mice, rather than just exposure to male urine, led to heightened activity in stress-related brain regions, including the lateral septum (LS), along with significantly increased levels of the stress hormone adrenocorticotropic hormone (ACTH) in the plasma.

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Key Findings of the Study

1. The Critical Role of ANCV1R in Pheromone Detection ANCV1R is indispensable for enhancing VNO sensory neurons’ responsiveness to multiple pheromone cues. Its absence led to reduced VNO responses to male urine, ESP1 (Exocrine Gland-Secreting Peptide 1), and other sexual pheromones.

2. Sexual Behavior Disorders ANCV1R−/− females failed to accurately detect and respond to male-specific chemical signals, resulting in a loss of attraction to males and abnormal rejection behaviors.

3. Amplified Stress Responses Physical interactions with males significantly activated neural activity in stress-related brain regions like the LS and MeA, leading to elevated systemic stress hormone (ACTH) levels in ANCV1R−/− females.

4. Altered Central Neural Feedback Reduced VNO pheromone input potentially altered feedback mechanisms, amplifying stress-related neural network activation during male-female interactions.

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Significance and Limitations

Scientific Significance

1. This study is the first to reveal the essential role of the highly conserved ANCV1R gene in regulating pheromone perception and sexual behavior in mice, offering key insights into mammalian chemical communication and its evolutionary mechanisms.
2. It underscores the importance of specific molecular elements in the pheromone system for enabling gender recognition and reproductive success in mammals.

Potential Applications

The findings provide groundwork for potential therapies targeting behavioral disorders, including social phobias and gender recognition impairments, by modulating pheromone perception mechanisms.

Limitations

1. The precise molecular mechanisms underlying the reduced sensory sensitivities in ANCV1R-deficient VSNs remain undefined.
2. Although the study focuses on mice, its applicability to other species with functional VNOs requires further verification.

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Conclusion

ANCV1R is a pivotal component of the murine pheromone detection system. Its deletion diminishes the capability of female mice to recognize male-specific chemical cues, impairing sexual behavior and triggering heightened stress responses. These findings highlight the indispensable role of ANCV1R in ensuring optimal social and reproductive interactions in terrestrial vertebrates, while providing an evolutionary perspective on pheromone signaling mechanisms.