Neuroanatomical and gene expression features of the rabbit accessory olfactory system. Implications of pheromone communication in reproductive behaviour and animal physiology

INTERNATIONAL DOCTORAL SCHOOL OF THE USC Paula Rodríguez Villamayor PhD Thesis Neuroanatomical and gene expression features of the rabbit accessory olfactory system. Implications of pheromone communication in reproductive behaviour and animal physiology. Doctoral Programme in Basic and Applied Research in Veterinary Sciences Lugo, 2023 TESE DE DOUTORAMENTO NEUROANATOMICAL AND GENE EXPRESSION FEATURES OF THE RABBIT ACCESSORY OLFACTORY SYSTEM. IMPLICATIONS OF PHEROMONE COMMUNICATION IN REPRODUCTIVE BEHAVIOUR AND ANIMAL PHYSIOLOGY Paula Rodríguez Villamayor ESCOLA DE DOUTORAMENTO INTERNACIONAL DA UNIVERSIDADE DE SANTIAGO DE COMPOSTELA PROGRAMA DE DOUTORAMENTO EN INVESTIGACIÓN BÁSICA E APLICADA ÁS CIENCIAS CLÍNICAS VETERINARIAS LUGO ANO 2023 DECLARACIÓN DO AUTOR/A DA TESE D./Dna. Paula Rodríguez Villamayor Título da tese: Neuroanatomical and gene expression features of the rabbit accessory olfactory system. Implications of pheromone communication in reproductive behaviour and animal physiology Presento a miña tese, seguindo o procedemento axeitado ao Regulamento, e declaro que: 1) A tese abarca os resultados da elaboración do meu traballo. 2) De ser o caso, na tese faise referencia ás colaboracións que tivo este traballo. 3) Confirmo que a tese non incorre en ningún tipo de plaxio doutros autores nin de traballos presentados por min para a obtención doutros títulos. 4) A tese é a versión definitiva presentada para a súa defensa e a versión impresa coincide coa presentada en formato electrónico E comprométome a presentar o Compromiso Documental de Supervisión no caso de que o orixinal non estea na Escola. En Lugo, de novembro de 2022 Fdo: Paula Rodríguez AUTORIZACIÓN DO DIRECTOR / TITOR DA TESE Neuroanatomical and gene expression features of the rabbit accessory olfactory system. Implications of pheromone communication in reproductive behaviour and animal physiology D. Paulino Martínez Portela (director) D. Pablo Sánchez Quinteiro (director) D. José Manuel Cifuentes Martínez (titor) INFORMAN: Que a presente tese, correspóndese co traballo realizado por Dna Paula Rodríguez Villamayor, baixo a nosa dirección/titorización, e a utorizamos a súa presentación, considerando que reúne os r equisitos esixidos no R egulamento de Estudos de Doutoramento da USC, e que como directores/titor desta non incorren nas causas de abstención establecidas na Lei 40/2015. De acordo co indicado no Regulamento de Estudos de Doutoramento, declaramos tamén que a presente tese de doutoramento é idónea para ser defendida en base á modalidade de COMPENDIO DE PUBLICACIÓNS, nos que a participación da doutoranda foi decisiva para a súa elaboración e as publicacións se axustan ao Plan de Investigación. En Lugo, de novembro de 2022 Fdo: Paulino Martínez Fdo: Pablo Sánchez Fdo: José Manuel Cifuentes Eu Paula Rodríguez Villamayor, declaro que esta tese de doutoramento non presenta conflictos de interés. En Lugo, de novembro de 2022 Fdo: Paula Rodríguez Published in: Animals (2022) 12:308. doi: https://doi.org/10.3390/ani12030308. Q1 in the category Agriculture, dairy & Animal science. Impact factor: 3.231. Contribution: Methodological and experimental design. Participation in the field study. Performing data analysis. Writing the original draft. Writing, reviewing and editing. Funding acquisition. Confict of interest: The authors declare that there is no conflict of interest. Copyright © 2022 Villamayor PR, Gullón J, Yáñez U, Sánchez M, Quinteiro P, Sánchez- Martínez P, Quintela L. This is an open access article distributed under the Creative Commons Attribution licence (CC-BY). No special permission is required to reuse all or part of article published by MDPI, including figures and tables. For articles published under an open access Creative Common CC BY license, any part of the article may be reused without permission provided that the original article is clearly cited (see Annex V). PhD chapter Journal IF 5-year IF Quartile Rank ISS N I Journal of Anatomy 2.479 (2017) 2.789 Anatomy & Morphology (Q1) 4/21 0021 - 8782 II Brain Structure and Function 3.622 (2018) 4.019 Anatomy & Morphology (Q1, D1) 1/21 1863 - 2653 III Genomics 6.205 (2019) 4.149 Genetics & heredity (Q1) 18/178 0888 - 7543 IV Frontiers in Molecular Neuroscience 6.261 (2021) 6.187 Neuroscience (Q1) 54/274 1662 - 5099 V Animals 3.231 (2021) 3.312 Veterinary Sciences (Q1) 16/144 2076 - 2615 ABBREVIATIONS Abbreviations . 2MB2: 2-methyl-but-2-enal. Also referred as MP in chapter II Aas: Amino acids AEMPS: Spanish agency of medicines and medical devices AI: Artificial insemination Ano: Anoctamin AOB: Accesory olfactory bulb AOS: Accessory olfactory system APHR: Aphrodisin AQPs: Aquaporins Arg: Arginase Bn: bouin liquid Bpifb3: BPI Fold Containing Family B Member 3 BSI-B4: Bandeiraea simplicifolia CB: Calbindin CCK: Cholecystokinin CF: Combined female CM: Combined male CNS: Central nervous system CR: Calretinin DAG: Diacylglycerol DE: Differentially expressed DEGs: Differentially expressed genes Dlk1: Delta like non-canonical Notch ligand 1 Dll4: Delta-like 4 signaling gene ECL: External cellular layer EPL: External plexiform layer ESP1: Exocrine glandsecreting peptide 1 ESPs: Exocrine secretory peptides ESR: Estrogen Esr: Estrogen receptor EU: European Union FC: Fold change FDR: False discovery rate FEL1A: Major allergen I polypeptide chain 1 F-F: Female-female; femiafemia FISH: Fluorescence in situ hybridization FLP: Female-specific lacrimal gland protein FPRs: Formyl peptide receptors Fr: buffered formalin Fshr: Follicle-stimulating hormone receptor GAP-43: Growth-associated protein 43 GFAP: Glial fibrillary acidic protein GG: Grüneberg ganglion GHRHR: Growth hormonereleasing hormone receptor GlL: Glomerular layer GLS: Glutaminase GnRH: Gonadotropin releasing hormone GO: Gene ontology GPA1: Alpha subunit of the heterotrimeric guanine nucleotide-binding G protein GrL: Granular layer Gαi2: G protein, subunit αi2 Gαo: G protein, subunit αo H2-Mv: A multigene family of non-classical class I major histocompatibility complex (MHC) genes ICL: Internal cellular layer IN: Interneurons IP3: Inositol 1,4,5- triphosphate IPL: Internal plexiform layer KEGG: Kyoto Encyclopedia of Genes and Genomes KO: Knockout LCN: Lipocalins LCN1_B: Lipocalin 1B LCN2: Lipocalin 2 LEA: Lycopersicon esculentum Lhcgr: Luteinizing hormone/choriogonadotropin receptor LOT: Lateral olfactory tract M&M: Material and Methods section MAP-2: Microtubule associated protein 2 MCL: Mitral cell layer MeA: Medial amygdala MHC: Major histocompatibility complex MOB: Main olfactory bulb MOE: Main olfactory epithelium MOS: Main olfactory system MP: Mammary pheromone MSP: Male-specific submandibular salivary gland proteins Mup4: Major urinary protein 4 MUPs: Major urinary proteins MYA: Millions of years ago NC: Nasal cavity NCBI: National Center for Biotechnology Information OB: Olfactory bulb OC: Oral cavity OMP: Olfactory marker protein ORs: Olfactory receptors Oxtr: Oxytocin receptor PAS: Periodic acid-Schiff PB: Phosphate buffer PBS: Phosphate buffered saline PCA: Principal component analyses PE: Paired-end Pgr: Progesterone receptor Pgrmc: Progesterone receptors membrane component PLC: Phospholipase C Pp: palatine process of the incisive bone PRG: Progesterone RD: Real Decreto RIN: RNA integrity number RNAseq: RNA sequencing RT-PCR: Reverse transcription polymerase chain reaction SBT: 2-sec-butyl-4,5- dihydrothiazole SF: Separated female SLC1A1: Excitatory amino acid transporter 3 SM: Separated male SO: Masera or septal organ SRA: Short Read Archive TPM: Transcripts Per Million TRPC: Transient receptor potential channel Trpc2: Transient receptor potential channel 2 UEA-I: Ulex europaeus V1Rs: Vomeronasal type 1 receptors (also called VR1 in chapter I) V2Rs: Vomeronasal type 2 receptors (also called VR2 in chapter I) VMH: Ventromedial hypothalamus VNL: Nerve layer VNO: Vomeronasal organ VNS: Vomeronasal system VRs: Vomeronasal receptors VSNs: Vomeronasal sensory neurons INDEX 3.5 Differential expression of the vomeronasal organ between females and males .......................................................................................... 217 3.6 Differential expression of the vomeronasal organ between juveniles and adults .......................................................................... 218 4. DISCUSSION ....................................................................................... 221 4.1 The unique gene repertoire of the rabbit vomeronasal organ ..... 221 4.1.1 Vomeronasal-type receptors ............................................... 222 4.1.2 Formyl peptide receptors.................................................... 224 4.1.3 Transient receptor potential channels ................................. 225 4.1.4 Major urinary proteins. Mup4 in the rabbit nasal mucosa . 226 4.2 VNO-mediated reproductive behaviour does not depend on VNO transcriptome sex differences ........................................................... 227 4.3 Insights into vomeronasal connectivity ...................................... 230 CONCLUDING REMARKS ........................................................................ 232 ACKNOWLEDGEMENTS .......................................................................... 232 SUPPLEMENTARY MATERIAL ................................................................ 233 CHAPTER IV: Sex separation unveils the functional plasticity of the vomeronasal organ in rabbits .................................................. 235 ABSTRACT .......................................................................................... 237 1. INTRODUCTION .................................................................................. 239 2. MATERIAL AND METHODS ................................................................ 242 2.1 Experimental design ................................................................... 242 2.2 Sampling .................................................................................... 244 2.3 Transcriptomic Analysis ............................................................ 244 3. RESULTS ........................................................................................... 246 3.1 Sex-separation induces sex-specific differences in gene expression of male and female rabbits ................................................................ 246 3.2 VNO transcriptome changes depending on environmental conditions in a sexspecific manner ................................................. 248 3.3 Vomeronasal receptor repertoire undergoes significant downregulation in adult female VNO under the sex-separation condition 249 3.4 Environmental modulation triggers differential expression of VNO genes involved in reproduction and sexual behaviour, supporting its plastic capacity to ensure individual survival ................................... 254 3.5 Environmental changes modulate VNO functional activity, thus adding complexity and flexibility to the VNO sensory code ............ 256 3.6 VNO as a first barrier in the detection of external stimuli, including harmful pathogens, and its relationship with the immune system ............................................................................................... 259 4. DISCUSSION ........................................................................................ 261 4.1 VNO and VR repertoires show species-specific and environmentally modulated expression: a comparison between rabbits and mice ............................................................................................ 261 4.2 The lipocalin aphrodisin is up-regulated in sex-separated adult male VNO ......................................................................................... 264 4.3 Sex-steroid receptors are modulated by the environment and may arise as a new type of VNO pheromone receptors ............................ 265 4.4 A potential close relationship between the VNO and female puberty .............................................................................................. 267 CONCLUDING REMARKS ......................................................................... 269 ACKNOWLEDGEMENTS ........................................................................... 269 SUPPLEMENTARY MATERIAL .................................................................. 269 CHAPTER V: Assessment of biostimulation methods based on chemical communication in female doe reproduction ................. 283 ABSTRACT .......................................................................................... 285 1. INTRODUCTION .................................................................................. 287 2. MATERIALS AND METHODS ............................................................... 289 2.1 Animals ...................................................................................... 289 2.2 Sample collection ....................................................................... 290 2.3 Semen processing and artificial insemination ............................ 290 2.4 Reproductive managemenent ..................................................... 290 2.5 Experimental design ................................................................... 291 2.6 Statistical analysis ...................................................................... 292 3. RESULTS .......................................................................................... 293 3.1 Fertility ....................................................................................... 294 3.2 Prolificacy .................................................................................. 296 3.3 Receptivity ................................................................................. 297 4. DISCUSSION ................................................................................... 299 4.1 Social interaction seems not influencing reproductive physiology in farm female doe............................................................................ 300 4.2 Urine as a potential source of sex pheromones in female doe reproduction ..................................................................................... 301 4.3 Seminal plasma might arise as a new source of pheromones .... .301 4.4 Practical considerations when assessing biostimulation methods 302 4.5 Could biostimulation methods reduce or replace hormonal treatments? ....................................................................................... 304 CONCLUDING REMARKS ....................................................................... 305 ACKNOWLEDGMENTS ................................................................................. 305 DISCUSSION .................................................................................. 307 1. IMPORTANCE OF AN INTEGRATIVE APPROACH IN CHEMOCOMMUNICATION RESEARCH ................................................ 309 2. COMPLEXITY OF VOMERONASAL CHEMORECEPTORS ........................ 313 2.1 V1R and V2R receptors .............................................................. 314 2.1.1 Extranasal expression of VRs ............................................. 317 2.2 FPRs ............................................................................................ 318 2.3 Sex-steroid receptors ................................................................... 319 3. IMPLEMENTATION OF PHEROMONES IN THE MARKET ........................ 321 CONCLUDING REMARKS AND FURTHER STEPS ............. 327 CONCLUSIONS ............................................................................. 329 REFERENCES ............................................................................... 335 ANNEX I ......................................................................................... 409 ANNEX II ........................................................................................ 421 ANNEX III ...................................................................................... 427 ANNEX IV ....................................................................................... 431 ANNEX V......................................................................................... 439 ANNEX VI ....................................................................................... 441 All figures included in this manuscript are original and elaborated by Paula Rodríguez Villamayor. Some figures were redrawed based on the content of referenced research articles and this is indicated in the corresponding figure footnotes. 51 SUMMARY Pheromone communication is involved in fundamental innate sociosexual behaviors such as mating and fighting, that are essential for animal reproduction and survival. Pheromonal cues are mainly perceived by the accessory olfactory or vomeronasal system (VNS), specifically by sensory neurons of the vomeronasal organ (VNO) which in turn send their inputs to the accessory olfactory bulb (AOB). The VNS greatly varies across evolution, especially at structural and genomic levels, to be able to face a high range of species-specific behaviours (i. e. different reproductive systems, dominances, etc.). Most VNS studies have been done in mice, which tended to extrapolate data to close phylogenetic species such as rabbits. However, due to the high specie-specificity of the VNS, this may lead to mistaken conclusions and should always be avoided; accordingly, each species should be studied independently and from a comparative point of view. Additionally, the rabbit -Oryctolagus cuniculusis considered a unique model for studying chemocommunication due to the discovery of the rabbit mammary pheromone. However, the rabbit VNS remains unexplored, especially at morphological, genetic and molecular levels. On the other hand, the study of pheromone communication could have a translational application into animal production (i. e. pheromones could be used as natural substances to improve reproductive parameters and animal welfare, which in turn may lead to a reduction of the use of hormones and antibiotics), and also into other industries such as pets (i. e. by reducing stress levels), or controlling endangered species or pests. Accordingly, it would be smart addressing research studies in species in which results could be PAULA RODRÍGUEZ VILLAMAYOR 52 directly transferred to the industrial sector. Rabbits are especially interesting because they are farmed species, easy to work with (small, short reproductive cycle) and more recently they have also become pets. Therefore, investigations into rabbit pheromone communication could be used for further translational work aimed at implementing pheromones in rabbit market. In this work, we aimed at characterizing the neuroanatomical and genomic features of the rabbit VNS as well as evaluating certain pheromone-mediated behavioral and reproductive parameters in rabbit farms. First, we studied the structural and morphofunctional properties of the VNO and the AOB. To do so, dissection, microdissection, histological techniques – histochemistry and immunohistochemistry–, and morphometry were employed. We determined that the rabbit VNO is highly developed and equiped to develop an effective chemocommunication. We also found that the AOB is sexually dimorphic and show a complex structure in terms of layer organization, neurochemical properties and cell types. We then explored the VNO at transcriptomic level to gain insights into their functional rationale. Specifically, an analysis of gene expression was carried out under different conditions – prepubertal, pubertal, male and female– by using RNAseq. The VNO gene expression did not vary between sexes but showed sharp differences between juvenile and adult rabbits. Besides the comprehensive characterization of the rabbit VNO transcriptome, including expression of vomeronasal receptors as well as many genes involved in reproduction, immunity and functional activity, we have also determined its plasticity –via transcriptional modulation–. To do so, we exposed rabbits to different environmental conditions (sexcombined vs sex-separated) and analysed their VNO gene expression comparatively. We found sex- and stagespecific gene expression differences upon such environmental conditions. Importantly, the vomeronasal receptors VRs showed significant down- and upregulated expressed genes in sex-separated adult female and sexseparated juvenile female, respectively. Similarly, genes involved in reproduction, immunity and functional activity were also found to be highly plastic in terms of their gene expression patterns. Finally, a first approach into translational studies was carried out directly in rabbit SUMMARY 53 farms. We developed a protocol which consisted of exposing female rabbits to either urine or seminal plasma as source of pheromones followed by an evaluation of their reproductive parameters compared to a control. Although we did not find any improvement in the female reproductive parameters, our protocol might be further used to try on other biological compounds such as extracts of exocrine glands, and determine their potential role in pheromone biostimulation. All in all, this study shows that pheromone communication and vomeronasal chemoperception is highly complex and should always be approached species-specifically and from a multidisciplinar perspective. Our data provide extensive knowledge into rabbit chemocommunication, specifically at anatomical, transcriptomic, reproductive and behavioural levels, which provide strong foundation for further translational studies which aim at implementing the use of pheromones in rabbit farms. Keywords: rabbit chemocommunication, vomeronasal system, vomeronasal organ plasticity, accessory olfactory bulb, sexual dimorphism, immunohistochemistry, RNAseq, pheromone biostimulation, reproduction, specie-specificity, multidisciplinar approach. GENERAL INTRODUCTION PAULA RODRÍGUEZ VILLAMAYOR 62 Odor: Refers to the detection and discrimination of all different chemical molecules (odorants) by the olfactory system (Wyatt, 2014a) Smell: Odors that are ‘consciously perceived’. They are mainly detected through the MOS. Semiochemicals/social odor signals: Complex specialized odors, also called chemosensory cues 3 , released by any living organism to the environment and perceived by others, providing information about the external world. They are mainly perceived by the VNS and act as key players in chemical communication (Wyatt, 2014a). They can be classified according to how they are released vs received among individuals (Figure 2): A) Chemical stimuli exchanged between members of the same species: - Pheromones (greek ‘pherein’) are specific olfactory cues/chemical stimuli released by one individual and perceived by another individual of the same species, triggering a behavioural or physiological response in the receiver (Wyatt, 2014b; Bakker and Leinders-Zufall, 2016). - Signature mixtures/instinctive cues: Subset of variable molecules (not pheromones) from the chemical profile of a conspecific that are learnt by the receiver for distinguishing individuals or colonies (Wyatt, 2014b). - Scent marks: Complex composition of chemical molecules, including the major histocompatibility complex (MHC) and major urinary proteins (MUPs), that provide genetic information about individuals regarding species, sex, individual identity, and metabolic information (social dominance and reproductive and health status) (Nevison et al., 2003; Arakawa et al., 2008). They originate from multiple sources, being urine and anal gland secretion the most common ones (Roberts, 2007) and their chemical composition 3 Semiochemicals are usually a mix of various molecules: (1) variable molecules, meaning those unique from each individual and (2) pheromones that are shared among groups of individuals. Altogether, they represent the chemical profile or chemical signature of an individual (Wyatt, 2014a). GENERAL INTRODUCTION 63 is thought to include both pheromones and other general odours (Johnson, 1973). The form of olfactory social communication between conspecifics mediated by scent marks (Melo et al., 2008) is defined as scent-marking 4 . B) Chemical stimuli exchanged between members of different species (Wyatt, 2014b; Bakker and Leinders-Zufall, 2016). These are called allelochemicals and they can be divided in: - Allomones, when they benefit the emitter. - Kairomones, when they benefit the receiver. - Synomones, when they benefit both the receiver and the emmiter. Figure 2. Diagram showing the different types of chemosensory cues involved in chemical communicaction. An example of ‘species-wide’ pheromone signals could be male sex pheromones, produced by any dominant male. Signature mixtures involve the process of learning (i.e. recognition of siblings) based on unique chemical profiles for each individual, and thus there is no species-wide molecule(s) to find. Modified from Wyatt, 2014b. 4 The most common scent-marking behaviour is territorial marking (between conspecifics), closely related to dominance, but it is also used as an alarm signal and as a threat to conspecifics as well as to other species. For instance, gland secretions may be deposited as a response to a predator odour with the effect of signaling alarm to conspecifics (Arakawa et al., 2008). This behaviour facilitates maintenance of a territory, reproductive competition, and sexual advertisement (Brown, 1979; Wolff, 2004). CHEMICAL COMMUNICATION (also called semiochemicals, social odor signals) Signature mixtures Pheromones Interaction between members of the same species Species-wide signals Allelochemicals Synomones Benefit the emitter Variable molecules, specific of each individual Interaction between members of different species Chemical profile / chemical signature Kairomones Benefit both the emitter and the receiver Benefit the receiver Allomones CHEMOSENSORY CUES PAULA RODRÍGUEZ VILLAMAYOR 64 In particular, pheromones are classified into different types based on the responses they trigger as well as on their chemical nature. They were first described by Karlson and Lüscher (1959) as chemical signals released by one individual to the environment and perceived by another individual of the same species, causing a physiological or a specific behavioural response in the receiver. They suggested the term would apply to chemical signals in all types of animals from invertebrates to fish to terrestrial mammals, and they could be single molecules or a mix of various molecules – even different species could share some molecules, probably causing different responses. Pheromones can be classified according to the response they trigger in the receiver (Wyatt, 2014a) (Figure 3): - Primer pheromones: They trigger a physiological response in the receiver, which is slow (not immediate) and usually modulated by the endocrine system (i.e. Early termination of pregnancy, or pregnancy block when a recently mated female is housed with a strange male, known as Bruce effect (Bruce 1959; 1960)). - Releaser pheromones: They trigger an immediate behavioural response upon reception (i.e. Nipple-search and suckling behaviour by rabbit kits, elicited by the rabbit mammary pheromone (2MB2; 2-methylbut-2-enal) (Schaal et al., 2003)). Vomeronasal sensory neurons NEUROCHEMESTRY Primer effect Releaser effect BEHAVIOURAL RESPONSE (immediate; direct) PHYSIOLOGICAL RESPONSE (slow; indirect) AOB stimulation Pheromones CNS (higher centers stimulation) GENERAL INTRODUCTION 65 Figure 3. Diagram showing how pheromones stimulate the VNS. Pheromones stimulate vomeronasal sensory neurons of the VNO, which send electrical inputs to the AOB and in turn prompt either direct behavioural responses (reseaser effect) or indirect physiological responses via stimulation of hormone secretion (primer effect). The same pheromone compound can act as primer and releaser at same time or in different contexts (Wyatt, 2009). Modified from Wyatt, 2014a. As previously mentioned, classical studies suggested that most pheromones are nonvolatile, activate the VNO, specifically VSNs, and trigger innate and stereotyped social behaviours and neuroendocrine release (Connor 1972; O’Connell and Meredith, 1984). However, this definition may be too restrictive and cannot explain all the behaviours induced by pheromones. In fact, Halpern (1987) already pointed out that the separation into volatile and nonvolatile compounds may be too simplistic. Accordingly, in the last decades, researchers have proven that the chemical nature of pheromones is highly diverse, and almost any type of chemical molecule (simple or mixed) has the potential to be a pheromone as long as they trigger the appropriate response in the receiver. Contrary to traditional thoughts, they can be either volatile or nonvolatile depending on the medium they are released (Brennan and Keverne, 2004). Additionally, pheromones can activate the VNO and also the main olfactory epithelium (MOE) of the MOS. Indeed, Halpern (1987) already argued that the MOS and the VNS may interact to yield specific responses to chemical signals, and traditional behavioural and neuroscience studies had always suggested that mammals use the VNS and the MOS, either independently or both working together, to perceive odors and semiochemicals, depending on species, signals, and previous experience. Importantly, inputs from the VNS and the MOS are integrated at higher centers in the brain (i.e. amygdala – see section 2.3 ‘Higher centers: limbic system and hypothalamus’ for detail) (Baxi et al., 2006; Meredith 1998; Wyatt, 2014a). Finally, pheromone effects can be altered by context –their responses are not as hardwired as previously thought– (Stowers and Marton, 2005). Therefore, pheromones are powerful signals that influence individuals’ behaviour, yet pheromone responses are not always stereotyped. Recent molecular and genetics advances have PAULA RODRÍGUEZ VILLAMAYOR 66 shown that variability in pheromone responses among individuals may be modulated at different levels of pheromone-processing circuitry, and in particular in the vomeronasal system –from the peripheral (VSNs) to higher centers in the central nervous system (AOB and vomeronasal amygdala)–. Studying the molecular mechanisms underlying variability in pheromone responses –i.e. VNS plasticity (see section ‘3. Plasticity of the vomeronasal system’ for detail)– should provide a needed framework for deciphering how pheromonal outputs are flexibly shaped into neural circuits and alter behaviour (Stowers and Liberles, 2016). 1.1.1.2 Chemosensory receptors of the olfactory system In mammals, the two major olfactory subsystems, the MOS and the VNS, are provided with OSNs and VSNs respectively, each with their own sets of receptors. Identifying such receptors as well as their mechanisms of action is a needed first step to further understand how this sensory system works. It was not until the nineties that a first large family of olfactory receptors –olfactory receptors (OR) in the MOE– was discovered by Buck and Axel (1991), a study which represents a big breakthrough in the field of olfaction and was awarded with the 2004 Nobel Prize for Physiology and Medicine. Soon after, researchers from Axel’s lab identified the vomeronasal receptor family (VRs) in the VNO (Dulac and Axel, 1995). Since then and thanks to the advent of new biotechnological and genomic techniques, great advances have been made in the field of olfaction and chemocommunication, especially regarding signal transduction, and the organization of the olfactory subsystems across evolution (Bakker and Leinders-Zufall, 2016; Brennan, 2018). In this part, we will briefly explain the different types of chemosensory receptors found in the MOS and VNS. In section ‘1.1.1.3 Evolution of the olfactory system in vertebrates’, the chemosensory receptors will be explained in an evolutionary context, and in section ‘2.1.2 Sensory receptors of the vomeronasal organ’ the vomeronasal receptors will be explained in detail. Across the mammalian chemosensory system, five types of GENERAL INTRODUCTION 67 chemosensory G-protein coupled receptors (GPCRs) have been identified so far: olfactory receptors (ORs) and trace amine associated receptors (TAARs) expressed in the MOE; and vomeronasal receptors (V1Rs and V2Rs) and formyl peptide receptors (FPRs), in the VNO. Additionally, a subset of VSNs in the VNO coexpresses V2R and H2- Mv, a subfamily of nine nonclassical class I major histocompatibility complex genes. Olfactory receptors (ORs) (Buck and Axel, 1991). They are the largest gene family in mammals, representing the 2-5% of the total genome (Barnes et al., 2020), and are located in OSNs in the MOE. Each mature OSN expresses only one OR –one receptor-one neuron– following a monoallelic fashion (Chess et al., 1994). Each OSN is able to respond to a high diversity of odorants; and conversely, each odorant can activate OSNs expressing different receptors (Malnic et al., 1999). This combinatorial code explains why we are able to detect more than one trillion of olfactory stimuli (Bushdid et al., 2014) with just a repertoire of several hundreds of ORs. Trace amine-associated receptors (TAARs) (Liberles and Buck, 2006). They have arisen as a second type of chemoreceptors located in the MOE. This family is expressed in a small subpopulation of OSNs and despite they do not co-express with ORs (Stowers and Logan, 2010), both share some features such as monoallelic expression and the one-neuron-one-receptor rule (Dewan et al., 2021). TAARs represent only the 1% (15 genes in mice and 6 in humans (Lindemann et al., 2005)) of all olfactory receptors (including ORs and TAARSs), but their evolutionary maintenance points to their fundamental role in olfactory function (Dewan et al., 2018). Despite their precise functionality is yet to be determined, these receptors are activated by chemical cues that show the hallmarks of pheromones (Liberles and Buck 2006). Some studies suggested that the MOS may be composed of two functional modules: one for the discrimination and associative learning of complex odours and the other for innate odour responses mediated by pheromones (Kobayakawa et al., 2007). Later studies suggested that ORs and TAARs could be the receptors specialized in PAULA RODRÍGUEZ VILLAMAYOR 68 the detection of learned vs innate responses, respectively (Johnson et al., 2012). Despite we know now that pheromone responses depend on context and therefore the terms ‘innate’ ‘hardwired’ and ‘sterotyped’ are not fully correct (Saraiva et al., 2016; Dewan et al., 2021), the involvement of TAARs in the detection of specific socioenvironmental cues makes them potential candidates for pheromone detection in the MOS (Liberles, 2009). The chemoreceptors of the VNO are explained in detail in section ‘2.1.2 Sensory receptors of the vomeronasal organ’. Briefly, there are two main types of vomeronasal receptors –V1R (Dulac and Axel, 1995) and V2R (Ryba and Tirindelli, 1997)–, which greatly vary across evolution, possibly to face adaptation to a continuous changing environment. Additionally, a second type of vomeronasal receptors – formyl peptide receptors (FPRs)– were identified (Rivière et al., 2009; Liberles et al., 2009) and appear to be exclusive of the rodent lineage. Finally, a subfamily of nine nonclassical class I major histocompatibility complex genes, called H2-Mv were identified as vomeronasal receptors that act jointly with V2R (Ishii et al., 2003). V1R and V2R receptors are differentially expressed in two VSN populations that also express the G-protein alpha-subunits: Gαi2 or Gαo, respectively (Jia and Halpern 1996). In contrast, FPRs coexpress with both Gαi2 and Gαo –though each individual receptor only expresses one G-protein– (Liberles et al., 2009; Riviere et al., 2009) (Figure 4). GENERAL INTRODUCTION 69 Figure 4. Schematic diagram illustrating the molecularly distinct apical (orange) and basal (green) layers of the vomeronasal sensory neuroepithelium –based on mice expression–. The three main vomeronasal receptors (V1Rs, V2Rs and FPRs) as well as their associated G protein coupled receptors (GPCR) are shown. Modified from Miras-Portugal, 2006. 1.1.1.3 Evolution of the olfactory system in vertebrates Olfaction is usually referred as one of the most 'primitive' sense in organisms – even bacteria are provided with a chemosensory system able to detect chemical molecules from the environment. Throughout evolution, different species evolved different receptors, structures and organs to be able to adapt to a continuously changing environment and ensure species survival. In Figure 5 and Table 1 we briefly summarize the distinct types of receptors and olfactory systems that vertebrates have evolved throughout evolution. Figure 5. Vertebrate phylogeny, showing the olfactory systems. MOS: main olfactory system. VNS: vomeronasal system. GG: grüneberg ganglion. MO: masera organ. PAULA RODRÍGUEZ VILLAMAYOR 70 Table 1. Olfactory systems and receptors of representative species from the different Vertebrate Superclasses. * amphibian V2Rs, 448 are pseudogenes –non-functional genes–. ** V1Rs and V2Rs repertoires greatly differ across species in mammals (see section ‘2.1.2 Sensory receptors of the vomeronasal organ’and Nei et al., 2008 for details). Phylogenetic Superclass MOS VNS ORs TAA Rs V1Rs V2Rs Refer. Agnata (lamprey) Yes VNS precur sor ~ 40 ~ 28 ~ 4 ~ 2 Beauséjo ur et al., 2022; Kowatsc hew and Korschin g, 2022) Gnathostomat a (shark) One unique ‘accessory system’ ~ 8 ~ 5 ~ 6 ~ 35 (Sharma et al., 2019) Osteichthyes (bony fishes) One system ~ 160 ~ 112 ~ 6 (ORAs) ~ 60 (OlfC) Nei et al., 2018; Villamay or et al., 2021b) Amphibia Yes Yes > 1000 7 22 697* Nei et al., 2008) Reptilia Yes Yes ~ 360- 1000 ~ 2 ~ 2-4 ~ 116- 430 Hogan et al., 2021) Aves Yes No > 200 ~ 3 (2) 0 0 Shi and Zhang, 2007; Vandewe ge et al., 2016 Mammalia Yes Yes ~ 800- 1200 ~20 ~0- 200** ~0- 200** Nei et al., 2008) The VNS was largely considered as an adaptation to terrestrial life, but this view is now questioned since VRs and even a sort of ‘VNS’ appeared before the water-to-land adaptation (i.e. lamprey, lungfish) (Ubeda-Abañón et al., 2011). A detailed description of the evolution of the olfactory system in vertebrates is beyond the scope of this introduction but useful references are added to Table 1. GENERAL INTRODUCTION 71 Mammals can be classified depending on their ability to smell in microsmatic (well-developed sense of smell (i.e. rodents, carnivores)), microsmatic (feebly developed sense of smell (i.e. primates)) or anosmatic (lacking the sense of smell (i.e. many aquatic mammals (Smith and Bhatnagar, 2004). In terrestrial mammals, there are two major and well-characterized nasal chemosensory subsystems –MOS and VNS–, containing a high diversity of gene families that encode MOE and VNO receptors. An additional level of complexity should be added to the organization of the sense of smell in mammals: 1) there are two other structures in the NC which were involved in detecting odours, namely the Masera or septal organ (SO) and the Grüneberg ganglion (GG), mainly studied in rodents (Salazar and Sánchez-Quinteiro, 2009; Munger et al., 2009). Since their axons project to specific areas of the MOB –SO to the ventromedial aspect of the MOB and GG to the caudal MOB, named the necklace glomeruli (Figure 1)–, some authors considered them as part of the MOS (Zimmerman and Munger, 2021). Even though their functional contributions are still unknown, there is evidence that both contribute to pheromone detection (Ma et al., 2003; Roppolo et al., 2006; Tirindelli et al., 2009); and 2) the MOE is further subdivided in various ‘subsystems’ which contain different olfactory sensory neuron subpopulations, each with their own receptors and projection-areas. Briefly, apart from the previously mentioned OSNs that express ORs and TAARs (called canonical OSNs), other non-canonical OSNs subpopulations are found in the MOE: OR37 neurons, GC-D+ OSNs and Trpc2+ OSNs (more extensive information can be found in Bader et al., 2012; Klein et al., 2015 for OR37 receptors; Greer et al., 2016 for GC-D+ OSNs; Omura and Mombaerts (2014) for Trpc2+ OSNs). Altogether, the mammalian sense of smell contains a diverse array of subsystems which are characterized based on the stimuli they respond to, the cell types and receptors they hold, their signal transduction mechanisms and the connections they establish to specific areas of the brain. This multifunctional task may allow for parallel processing of chemical cues, in which each subsystem probably serve a specific function therefore conveying different types of information from a single chemical cue. PAULA RODRÍGUEZ VILLAMAYOR 78 interaction, and simple comparisons of the V1R gene repertoire, even between closely related species, may lead to unexpected and fascinating findings, which ultimately would bring us one step closer to the understanding of social communication in mammals. 2.1.2.2 Vomeronasal type-2 receptors V2R receptors also belong to the seven-transmembrane domain GPCR family, but they share no sequence homology with V1Rs. Thus, V1Rs and V2Rs are generally considered to have evolved independently. V2R genes have been identified in vertebrates, and are supposed to be restricted, similar to V1Rs, to this subphylum (Rodriguez, 2008). In contrast to V1Rs, the number of functional V2Rs is not correlated with VNO complexity (Grus et al., 2007). The largest V2R gene repertoire is found in amphibians (see Table 1). In mammals such as mice and rats, 61 (updated to 122 by Ishii and Mombaerts, 2009) and 57 V2R genes with intact open reading frames have been reported, respectively (Yang et al., 2005); therefore, V2R repertoire in rodents appears to be smaller than the V1Rs repertoire. In other species such as opossum and platypus, 90 and 15 V2Rs have been found, respectively. Until recently it was thought that aryctiodactila did not posses V2Rs, but a few active V2R genes were found encoded in the genome of cattle, goats, sheep and pigs (Kondoh et al., 2022). Nonetheless, no functional V2R genes have been found in dogs, chimpanzee, macaque and humans (Grus et al., 2007; Young et al., 2007) (Table 2). V2Rs also have a large repertoire of pseudogenes in mice (148) and rats (111) (Yang et al., 2005). Platypus and opossum have 57 (Grus et al., 2007) and 79 (Young et al., 2007), respectively, and between 9-20 were found in cow, dogs and humans (Young et al., 2007) (Table 2). At molecular level, V2Rs differ from V1Rs by the presence of introns and the long N-terminal extracellular region, which is encoded by five exons –in contrast with V1Rs that only hold one exon– and likely reflects the ligand-binding site (Francia et al., 2015). Despite the vast majority of V2R genes were only expressed in the VNO, Ibarra-Soria et al., (2014) found expression of one V2R in GENERAL INTRODUCTION 79 the MOE, Vmn2r29, suggesting previous unrecognized mechanisms of pheromone detection in the MOE. Functionally, V2Rs have proven to detect high molecular weight non-volatile proteins and peptides (Roberts et al., 2010). In particular, the potential ligands for V2Rs include peptide pheromones such as exocrine gland-secreting peptide 1 (ESP1) (Kimoto et al., 2005), mouse urinary proteins (MUPs) (Chamero et al., 2007) and MHC peptides (Leinders-Zufall et al., 2004). Later studies suggested that V2Rs may be devoted to encode information about the identity of emitters (i.e. gender identity, heterospecifc cues such as predator vs non-predator), with clear behavioural significance (Isogai et al., 2011). At gene expression levels, both V1Rs and V2Rs showed variation across different mice strains. Although these differences are not equivalent to genetic differences at the nucleotide level, they are nonetheless important traits that can provide information about evolutionary divergence among mice strains (Duyck et al., 2017). Table 2. Number of V1Rs and V2Rs in different mammal species. Numbers in parenthesis indicate pseudogenes. Data obtained from Nei et al., 2008; Kondoh et al., 2022. Species V1Rs V2Rs Mice 187 (121) 121 (158) Rats 106 (66) 79 (142) Cattle 24 (8) 2 (11) Goats 9 (2) 1 (2) Sheep 16 (4) 1 (2) Pigs 16 (9) 10 (3) Dogs 8 (33) 0 (9) Chimpanzee 0 (116) 0 (17) Humans 5 (115) 0 (20) 2.1.2.3 Formyl-peptide receptors FPRs are seven transmembrane domain GPCR belonging to the rhodopsin-like superfamily and were first described in immune cells (Le et al., 2002). In mammals, fpr1 and fpr2 are believed to play an important role in innate immune response and are expressed by PAULA RODRÍGUEZ VILLAMAYOR 80 immune cells such as granulocytes and monocytes (He et al., 2013; Ackels et al., 2014). The evolutionary history of this immune family is highly complex (Silva et al., 2020); while a single copy of fpr1 has been described in mammals, fpr2 has undergone dynamic episodes of duplication events widely varying among species, that points towards the frequent neofunctionalization of this subfamily. In rodents, at two different times, an fpr coding sequence was integrated in a VR gene cluster, acquiring the regulatory elements of VRs and leading to their expression in the VNO. In other words, two gene shuffling events forced an immune pathogen sensor to transition into an olfactory chemoreceptor (Dietschi et al., 2017; Boillat et al., 2021). Consequently, in Rodentia, a third class of VNO receptors was found exclusively expressed in vomeronasal tissue and represents an expansion of the immune gene fpr2. VNO FPRs include 5 out of the 7 members of the formyl peptide receptor family (fprs) -Fpr-rs1 (also known as Fpr3), Fpr-rs3, Fpr-rs4, Fpr-rs6 and Fpr-rs7, (Rivière et al., 2009; Liberles et al., 2009). There is no evidence of co-expression of FPRs with V1Rs or V2Rs, and it has been argued that specific subsets of VSNs exclusively express FPRs in a monogenic manner (Liberles et al., 2009; Riviere et al., 2009). The ligand types that bind VNO FPRs remain largely unknown but they might play a role in VNO pathogen sensing (Bufe et al., 2012) and in mediating sickness conspecific avoidance (Boillat et al., 2015;Tirindelli, 2021; Dietschi et al., 2017). In fact, Bufe et al., (2019) recently found that the non-volatile bacterial MgrB peptide activates an FPR subtype (Fpr3) which is expressed in a subset of vomeronasal sensory neurons and which drives avoidance behaviour (Bufe et al., 2019). Although this represents the only functional study of any FPR in the VNO to date, FPRs constitute promising candidates for the detection of sickness-related cues. Therefore, VNO FPRs may have an important function at controlling the virulence of infections, placing this sensory organ as a potential interface between the external world and the immune system (Bufe et al., 2012, 2015, 2019). Finally, although most studies on FPRs have been restricted to rodents and primates (Dierschi et al., 2017; Yang and Shi, 2010), recent finding showed fpr2 paralogs in Chiroptera and Perissodactyla, GENERAL INTRODUCTION 81 though their putative expression in the VNO remains unknown (Silva et al., 2020). These results suggest that fpr2 expansions may have not been restricted to Rodentia, and further studies using high quality genome assembles will help deciphering the evolutionary history of this unusual genomic event. 2.1.2.4 H2-Mv receptors In mice, nine genes belonging to the family of nonclassical class I major histocompatibility complex (MHC) genes (H2- Mv), were found differentially expressed in subsets of basal VNO neurons (Ishii et al., 2003; Loconto et al., 2003; Ishii and Mombaerts 2008; Leinders-Zufall et al., 2009). These vomeronasal-specific genes were found co-expressed with V2R (Ishii et al., 2003). To our knowledge, no information of H2-Mv genes has been reported in other mammal species. Functionally, although dispensable to trigger physiological responses in the VNO neurons, H2-Mv genes contribute to ultrasensitive detection of pheromones by a subset of VSNs (Leinders-Zufall et al., 2014). However, their specific role in the VNO needs further investigation and in fact whether these molecules should be considered as pheromone receptors remains controversial (Ishii and Mombaerts 2008; Francia et al., 2014). 2.1.3 Signal transduction mechanisms VNO signal transduction is complex and not yet fully understood. Most studies have focused on the transduction mechanisms for V1Rs and V2Rs receptors, but new studies are now questioning the traditional view in which specific G proteins -Gαi2 and Gαowere thought to directly correlate with V1R and V2R expression. In general, in the VNO the signal is initiated by a pheromone-like ligand binding to one of two types of VNS-specific GPCRs, V1Rs or V2Rs. This binding modifies the receptor conformation, causing the release of the βγ complex of the heterotrimeric G proteins Gαi2 and Gαo, for V1Rs and V2Rs, respectively. This activates phospholipase PAULA RODRÍGUEZ VILLAMAYOR 82 C (PLC), which will increase levels of two secondary messengers, diacylglycerol (DAG) and inositol 1,4,5- triphosphate (IP3) (Minke and Cook, 2002). Both DAG and IP3 increase the intracellular calcium level -DAG by activating the Trpc2 channel allowing a depolarizing influx of Na+ and Ca2+ and IP3 by allowing the release of intracellular calcium stores- (Figure 7). Trpc2 appears to be the main transduction channel in V1R-expressing neurons but its role in V2R- expressing neurons is less clear (Francia et al., 2014). While some of the signal transduction molecules are common to other signal transduction pathways, three types of VNS genes (V1Rs, V2Rs, and trpc2) are known to function in the VNS-specific chemoreception. Figure 7. Transduction mechanisms of V1Rs and V2Rs. PLC: phospholipase C; DAG: diacylglycerol; IP3: inositol 1,4,5- triphosphate. Modified from Tirindelli et al., 2009. GENERAL INTRODUCTION 83 In some orders such as Rodentia and Didelphimorphia, VNO show segregated expression of specific G-protein coupled receptors: Gαo, expressed at the basal VNO neuroepithelium; and Gαi2, expressed at the apical part, (Halpern et al., 1998). However, not all mammalian VNOs have this segregated pattern (i.e. in fox Gia2 and Gαo were found intermingled in the VNO neuroepithelium (Ortiz-Leal et al., 2020). Furthermore, as previously mentioned, in some mammal species intact V2R genes have completely degenerated (see section ‘2.1.2.2 Vomeronasal type-2 receptors’), and consequently, they usually lack VNO Gαo expression. Accordingly, two types of VNO G expression patterns can be defined: 1) dichotomous expression of Gαo and Gαi2 in species with intact V1Rs and V2Rs such as mouse, rat, opossum (Jia and Halpern, 1996) and 2) exclusive expression of Gαi2 in species which lack V2Rs such as goat (Takigami et al., 2000), sheep (Salazar et al., 2007), dog (Salazar et al., 2013) and cat (Salazar and Sanchez-Quinteiro, 2011). Notwithstanding, recent studies have added an additional level of complexity to this organization: in fox, Gαo expression has been found in the VNO but no V2Rs (intact or pseudogenes) have been identified in the fox genome (Ortiz-Leal et al., 2020). The authors suggested that this would be the first case in mammals in which no V2Rs pseudogenes exist and point towards low quality fox genome assemblies, more than a real scientific fact. In any case, VSNs project their axons to the AOB glomerular layer (see Figure 8 and section ‘2.2.2 Functional circuitry of the AOB’). Species that express both types of G proteins –Gαi2 and Gαo– show an expression pattern restrictive to the anterior (Gαi2 expression) and posterior (Gαo expression) parts of the AOB (glomerular and nerve layers), respectively. If the species only expresses Gαi2, its expression covers the entire surface of the glomerular-nerve layers of the AOB. In fox, where Gαo expression was found in the VNO, such expression was lacking at the AOB level, suggesting that Gαo-mediated projections from the VNO could be reaching the MOB rather than the AOB (Ortiz-Leal et al., 2022). Additionally, results from our group (unpublished) indicate that rabbit fetus show strong patterns of Gαi2 and Gαo in both the VNO and the AOB, but the expression of V1Rs and V2Rs –analyzed by RNAseq– PAULA RODRÍGUEZ VILLAMAYOR 84 is almost absent. Finally, VNO FPRs have also proved to co-express with Gαi2 and Gαo. Specifically, Fpr-rs1 coexpresses Gαo and all the rest (Fprrs3,4,6,7) coexpresses Gαi2 (Liberles et al., 2009). In summary, these results show that although Gαi2 and Gαo usually show a correlated expression with V1Rs and V2Rs, respectively, they do not exclusively do; and that the interplay between the VNS and the MOS could be already established at the first step of the sensory processing. Though it seems clear that all of the mentioned elements (i.e. V1Rs, V2Rs, G proteins, Trpc2, etc.) contribute to the signal transduction machinery, the logic behind their expression appears to be highly complex and other yet unknown specie-specific G proteins and vomeronasal receptors may also be implicated in signal detection and transduction. 2.2 Accessory olfactory bulb VSNs axon bundles form the vomeronasal nerve, which enter the brain through the cribriform plate and target a specialized region usually located at the posterior dorsal part of the MOB -it seems to be embedded in it-; the AOB. The AOB is therefore the first neural integrative centre of the VNS. Although this structure was first described in the late 19th century by Gudden (1870) and it was one of the earliest cortical areas studied by classic neuroanatomist, its apparent absence in humans together with its extremely high complexity has hampered its investigation, especially at cytological and neural circuit levels. Both AOB and MOB share some similarities including their layered organization, classes of neuronal cell types and functional connectivity (Mohrhardt et al., 2018). Note, however, that both structures also show important differences with major physiological implications and therefore, extrapolation of the structural and functional principles from the MOB to the AOB should be taken with caution (Dulac and Wagner 2006; Stowers and Spehr 2015). The next part of the introduction will focus on the main features of the AOB, particularly in comparison to those of the MOB. GENERAL INTRODUCTION 85 2.2.1 Structure of the AOB The AOB is located at the anterior part of the encephalon and caudally to the MOB. Both MOB and AOB present a comparable structure in terms of layers and cell-types. Since the MOB has been more broadly studied than the AOB, the layering and cell-type nomenclature of the AOB have been an extension of that previously described in the MOB. Though this seems a practical and suitable approach, it has sometimes led to misunderstanding and lack of accuracy of some AOB specific features. In general, both MOB and AOB can be understood as elongated onions made up of different layers that are organized in concentric circles (Olude et al., 2014). Traditionally, the layers that form the MOB and, by extension, also the AOB are –from the outside to the inside of the bulb– 1) the nerve layer (VNL), 2) the glomerular layer (GlL), 3) the external plexiform layer (EPL), 4) the mitral cell layer, 5) the internal plexiform layer (IPL), and 6) the granular layer (GrL) (Allison, 1953) (Figure 8). The latter can be split into external and internal granular layer. PAULA RODRÍGUEZ VILLAMAYOR 86 Figure 8. Schematic representation of the VNS pathway from the environment to the VNO and to the AOB, showing the different AOB layers. The layers of the AOB and the different types of neurons are represented. VNO: vomeronasal organ; AOB: accessory olfactory bulb; ECL: external cellular layer; ICL: internal cellular layer; VNE: vomeronasal neuroepithelium; VNL: vomeronasal nerve layer; GlL: vomeronasal glomerular layer; EPL: external plexiform layer; MCL: mitral/cell layer; IPL: internal plexiform layer; eGrL: external granular cell layer; iGrL: internal granular layer; VSNs: vomeronasal sensory neurons. Modified from Galliano Lab at https://gallianolab.org/. Regarding cell types, the AOB is provided with excitatory principal cells (the MOB homologous are named as mitral/tufted cells) that receive input from OSNs and send output to higher brain centers. They are found in the MCL (also in the EPL in some cases) and can be subdivided according to their shape (i.e. rat AOB: large principal cells, round projecting cells and tufted cells (Larriva-Sahd, 2008). There are also inhibitory interneurons (IN): periglomerular cells in the GlL and granule cells in the GrL. Due to the high complexity of the system, even though advances have been made recently in terms of cell-types and specie-specific features, the current literature still maintain the traditional nomenclature as a reference, and very few studies face the variability and complexity of the bulbs. Importantly, Larriva-Sahd (2008) on his study of the rat AOB pointed towards the need of differentiating the AOB and MOB nomenclatures and proposed a new nomenclature to the AOB. His team proposed two new layers: the external cellular layer (ECL), containing the plexiform layers (EPL and IPL), the MCL, and the external part of the GrL; and the internal cellular layer (ICL), replacing the internal GrL (Figure 8). The AOB appears to retain the structural dichotomy observed in the VNO: the two main subsets of either Gαi2 or Gαo expressing neurons target two segregated regions in the VNL and GlL along its rostro-caudal axis. Gαi2 positive neurons are located in the rostral region of the AOB, whereas Gαo-positive are expressed in the posterior region of the AOB. Though this is usually correlated with the expression of V1Rs and V2Rs, respectively (Martinez Marcos, 2008), this is not always the case (see section ‘2.1.3 Signal GENERAL INTRODUCTION 87 transduction mechanisms’ for detail). Also, axons of FPR-rs3- expressing VSNs converge onto multiple (~8) glomeruli in a spatially restricted region of the rostral AOB, which does not overlap with V1Rs and V2Rs expressing VSNs (Dietschi et al., 2013; Mohrhardt et al., 2018). Phylogenetically, the AOB has been subjected to higher speciesspecific structural and variations than the MOB (Meisami and Bhatnagar 1998), especially in terms of AOB location, shape, size, cell-types specificity, morphological features and developmental patterns. AOB is well developed in rodents (Rodriguez et al., 1999), marsupials (Jia and Halpern 2004), and prosimians (Skeen and Hall, 1977). In some species such as dogs (Salazar et al., 2013), mink (Salazar et al., 1998) and some bats (Frahm and Bhatnagar, 1980), the AOB is present but appears to be poorly developed, and in some others such as African elephant (Ngwenya et al., 2011), West Indian manatee (Mackay-Sim et al., 1985) and human (Trotier et al., 2000), the AOB is absent or has not yet been identified. Additionally, regarding sexual dimorphism, previous studies on adult rats showed differences in the AOB volume of males and females (Segovia et al., 1984), as well as in the density of new-born cells at the anterior part of the AOB (Peretto et al., 2001), which appears to be specifically linked to discriminating reproductive-related chemosensory stimuli (Dulac and Torello, 2003; Oboti et al., 2009). 2.2.2 Functional circuitry of the AOB VSNs project their axons directly to the AOB, which is the initial site of the adult mammalian brain where vomeronasal sensory information is first processed. VSNs expressing VRs project their axons to activate multiple neuropil sac-filled structures, known as glomeruli which reside in the GlL of the AOB. Glomeruli are tightly clustered and sparsely surrounded by periglomerular IN. Contrary to the MOB, periglomerular cells do not define a clear boundary around each glomeruli but rather establish a fine line which separates the GlL from the MCL (Tirindelli et al., 2009). An important difference between the AOB and the MOB is that while each OSNs expressing PAULA RODRÍGUEZ VILLAMAYOR 94 3.1.1 A lifetime of neurogenesis in VSNs Neurogenesis represents another key weapon in the adult nervous system’s plasticity armoury for dealing with a constantly changing world (Lledo et al., 2006). Briefly, though neurogenesis was largely linked to embryonic and early postnatal stages in vertebrates, it is nowadays well-known that newly generated neurons continue throughout life in specific regions of the nervous system. In particular, three main neurogenic niches have been identified: 1) subgranular zone –which send neurons to the dentate gyrus of the hippocampus –; 2) subventricular zone –which send interneurons (mostly granular but also periglomerular cells) to the main and accessory olfactory bulb–; and 3) olfactory and vomeronasal neuroepithelia –which generate new excitatory olfactory and vomeronasal sensory neurons whose axons connect to the main and accessory olfactory bulb, respectively (Kageyama et al., 2012; Brann and Firestein, 2014). The two latter supply new neurons to the first level structures of the olfactory system –sensory neurons and olfactory bulbs–. Notably, adult neurogenesis in the olfactory system, and in particular in the olfactory bulb, is of paramount for sensory discrimination, odour-based learning, and reproductive social behaviours (Pignatelli and Belluzzi, 2010; Lledo and Valley, 2016). However, while the role of subventricular zone neurogenesis in learning seems clear (Moreno et al., 2014), the olfactory neuroepithelia remains understudied (Brann and Firestein, 2014). VSNs originate as stem cells in the marginal zones near the dorsal and ventral aspects of the VNO and then migrate horizontally along the basal zone where they differentiate into first immature and then mature functional VNO neurons. It is commonly known that VSNs undergo regeneration throughout life both physiologically and after injury (Martínez-Marcos et al., 2005; Brann and Firestein, 2014). Interestingly, though VSNs regenerative and proliferative capacity seems to decrease in natural aged animals (Mechin et al., 2021; Portalés et al., 2022), it remains robust in response to damage even with advanced age (Brann and Firestein, 2010). In contrast, aged hippocampus lacks of neuron regeneration following injury (Shetty et GENERAL INTRODUCTION 95 al., 2010). This places VSNs at the forefront of stem cell therapies, which may require isolation from an aged system, as the onset of most neurodegenerative diseases is during later life stages (Brann and Firestein, 2010). The functional rationale of VSNs regeneration is not clear, but it seems to be influenced by epigenetic effects (Xia et al., 2010). All in all, VSNs neurogenesis remains an underexplored yet promising field of study. Future work should aim at understanding the proliferative nature of these neural stem cells, especially in the context of their therapeutic implications (Brann and Firestein, 2010; 2014). Of note, even though OSNs neurogenesis is beyond the scope of this introduction (see in detail in Mackay-Sim et al., 2015), it follows a comparable pattern to that of VSNs. Overall, despite OSNs neurogenesis has been more broadly studied than VSNs neurogenesis, even with advance single-cell technologies (Hanchate et al., 2015; Durante et al., 2020), the regulation of olfactory neurogenesis is not completely understood in any of the two systems. 3.2 Plasticity of the AOB MOB and AOB are structures that display an important plasticity receiving olfactory axons, newborn interneurons and showing adaptive changes of the intrinsic and also the centrifugal afferents neurons (Diaz et al., 2017; Zhang and Meeks, 2020). One of the main plastic mechanisms of these systems arise from the incorporation of new local GABAergic inhibitory neurons –mostly granular but also periglomerular interneurons (Oboti and Peretto, 2014)– to the existing circuits throughout life (Oboti et al., 2009; Wu et al., 2020). However, less is known about their neurochemical profile and functional integration into the bulb, especially in the case of AOB, where information processing is poorly understood compared with the MOB (Oboti et al., 2009; Zhang and Meeks, 2020). The maturation and survival of newborn interneurons in the MOB depends on sensory inputs, as shown by olfactory enrichment and deprivation studies (Rochefort et al, 2002; Mandaironetal, 2006). Indeed, 24 h sensory deprivation –via naris occlusion– is sufficient to PAULA RODRÍGUEZ VILLAMAYOR 96 induce functional plasticity in a specific subtype of MOB interneurons (Galliano et al., 2018). Although renewing at a slower rate (Oboti et al., 2009), newborn cells in the AOB are likely to similarly contribute to VNS function. Sensory activity induced by chemical stimuli present in bodily secretions or urine increases the survival of newborn AOB interneurons (Oboti et al., 2009, 2011; Nunez-Parra et al., 2011). Interestingly, exposure to male-soiled bedding significantly increased the number of new neurons in the AOB of female mice (Oboti et al., 2009), pointing towards a key role of newborn interneurons –and in particular granule cells– in the regulation of reproductive and social behaviour (Peretto et al., 2014). Subsequent studies showed that mating behaviour originates long-term sensory memory for the pheromones of the stud male, and that this is directly linked to mitral cells plasticity and AOB sensitivity (Gao et al., 2017). Furthermore, male-male social interaction has proven to drive AOB inhibitory plasticity in which mitral cell activation is suppressed by increased excitability of newborn granule cell (Cansler et al., 2017); this sensory inhibition is critical for the regulation of behavioural responses to social chemosignals (Zuk et al., 2022). Therefore, AOB adult neurogenesis and mitral cell vs interneuron communication seems to be regulated by an experience-specific mechanism and may be functionally linked to the specific role of the VNS in pheromoneperception (Oboti et al., 2009), critical for animal physiology and behaviour (Zhang and Meeks, 2020). Finally, male pheromones have proven to be involved in regulating neurogenesis, not only in the AOB but also in the MOB and hippocampus (Mak et al., 2007). Further work should approach the molecular and circuit logic behind the AOB and the other neurogenic niches –MOB and hippocampus–, to understand how learning and social behaviours are impacted by newborn neurons. 4. RABBIT Rabbit represents a suitable model of chemocommunication in mammals. Since rabbits are farmed for animal production and also GENERAL INTRODUCTION 97 lately became pets, outcomes of our studies in rabbits will not only have impact in the research community but also set the basis to apply pheromones at industrial level, to enhance reproductive parameters and animal welfare. 4.1 Taxonomy Rabbits have been considered rodents until early 20th century. However, contrary to such popular belief, rabbits belong to the Order Lagomorpha and differ from rodents in several traits related to teeth - they have an extra pair of incisors- and other digestive and reproductive features. Lagomorpha and Rodentia have diverged from a common ancestor called Glires around 64.5 millions of years ago (57.3–73.3) (Rose et al., 2008). Brandt (1855) already considered Lagomorphs as a suborder within rodents, but it was not until 1912 when JW Gidley officially stated Lagomorpha as a separate Order. Lagomorpha is divided into 2 families comprising 92 species: Ochononidae (29 species; pikas) and Leporidae (63 species; rabbits and hares) (Ruedas et al., 2018). The European rabbit (wild or domestic rabbit) is the most well-known species of rabbit and the only one belonging to the genus Oryctolagus. Its complete taxonomic classification is as follows (ITIS, 2022): Domain: Eukaryota Kingdom: Metazoa Phylum: Chordata Subphylum: Vertebrata Class: Mammalia (Linnaeus, 1758) Order: Lagomorpha (Brandt, 1855) Family: Leporidae (Fischer, 1817) Genus: Oryctolagus (Lilijeborg, 1873) Species: Oryctolagus cuniculus (Linnaeus, 1758) – European Rabbit 4.2 Biology The European rabbit (Oryctolagus cuniculus) is native to the PAULA RODRÍGUEZ VILLAMAYOR 98 Iberian Peninsula, western France and Northwest Africa but it was successfully introduced in many countries across the world since medieval times, and its distribution is nowadays considered worldwide (Cooke, 2018). Their ear and eye are well-developed allowing early detection of predators. They also use a variety of olfactory signals / pheromones released by exocrine glands (i.e. chin gland for territory marking and mammary-gland for nipple-search behaviour) to communicate among themselves, and conduct information related to reproduction, dominances, maternal care, etc. (Melo and González-Mariscal, 2010). Rabbits are well-known for their reproductive capacity. Gestation lasts for about 30 days, and litters range between 6-10 kittens / litter. Females are of induced ovulation, which means that eggs are only released after copulation (Nowak, 1999; Vaughan et al., 2010). Rabbits have been raised commercially for meat, fur, as pets (Varga, 2014) and also for research purposes. In this latter, in addition of being a model of chemical communication studies (Schaal et al., 2003), (see section ‘4.3 Rabbit as a model of chemocommunication’ for detail), they are well-known for antibody production and also became an animal model to study human physiology and pathology such as human pregnancy, fetal development or osteoarthrosis (Banks, 1989; Esteves et al., 2018; Xu et al., 2021). 4.3 Rabbit as a model of chemocommunication To date, most VNS studies have been focused on laboratory rodents. Due to the phylogenetic proximity between Rodentia and Lagomorpha, studies have been tempting to wrongly extrapolate information form mice and rats to rabbits (Salazar and Sanchez- Quinteiro, 2009). As we stated throughout the introduction, the VNS greatly varies among species and even between strains and individuals, in terms of structure, function, and molecular and genomic features. Rabbits are considered one of the best models for studying chemocommunication in mammals (Schneider et al., 2018). This species remains as the only mammal species in which a mammary GENERAL INTRODUCTION 99 pheromone, the 2MB2, has been fully characterised (Schaal et al., 2003). MP is released by lactating females and triggers the nipplesucking reflex in rabbit neonates. Though previous studies pointed to the MOS as MP chemodector (Charra et al., 2013), receptors involved in its detection have still not been identified. Additionally, some behavioural studies have also pointed towards the importance of pheromone communication in rabbits (i.e. the rabbit ‘male effect’ points to an improvement of doe reproductive performance (El-Azzazi et al., 2017). The VNS ranks as the main pheromone detector system in mammals. However, it is a paradox that rabbits –considered a model of chemocommunication– lack of comprehensive VNS studies. Specifically, anatomical and physiological studies on the rabbit VNO and AOB have been scarce in the literature (see introductions of chapters I and II for more details). Also, rabbit VNO has not been included in most VNO phylogenetic studies and no functional genomic studies have been approached to assess gene expression (see introduction of chapter III). Therefore, behavioural studies lack of integrative approaches which consider the structural, functional and molecular rationale of pheromone-communication. Rabbit-specific new –OMIC technological studies at the level of the VNS are urgently needed, for example to decipher the rabbit VNO VR repertoire as well as to define its gene expression patterns. Farmed rabbits arise as a suitable model to study the structure and function of the VNS as well as to perform pheromone-like exposure trials. On the one hand, VNS studies in a more ‘natural’ environment (outside lab conditions) will provide a better picture of how this sensory system integrates external cues and internal states to instruct behaviour. On the other hand, results obtained could be directly translated into industry. Finally, rabbits are also a convenient species to work with because of its small size, easy handling, short reproductive cycle (1 month), and high fertility and prolificacy. 4.4 Cuniculture Rabbit farming or cuniculture is the agricultural practice of PAULA RODRÍGUEZ VILLAMAYOR 100 breeding rabbits as livestock, mainly to produce meat for human consumption. Rabbit meat is highly recommended, not only for its low fat content –similarly to other white meats– but also for its richness in highly-quality proteins, omega-3 fatty acids, vitamin B12 and minerals like calcium and potassium (Cullere and Zotte, 2018). 4.4.1 Current status of cuniculture The European Union (EU) is the second largest producer of rabbit meat worldwide, after China (EFSA, 2019). Cuniculture within the EU is mainly concentrated in three countries, representing the 83% of the total EU production: Spain (48.5 million of animals/year), France (29 million) and Italy (24.5 million) (European Commission, 2017). Therefore, Spain is considered an industrial reference in rabbit meat production throughout the European territory. Specifically, our country generates more than 50,000 tons of rabbit meat annually. Within the Spanish territory, Galicia occupies the second position in the ranking, just after Cataluña, with a production of almost 11,500 tons per year –data from 2018; there is still no available data of Galicia from 2019 onwards– (MAPAMA, 2021a; 2021b). In particular, the company COGAL (Rodeiro, Pontevedra), which has been actively collaborating with this PhD project, is responsible for more than 50% of the rabbit meat production in Galicia. Worldwide, from 2010 to 2016 rabbit meat market increased by 1.16%, from 1,224,186 to 1,428,085 tons, with China as the largest rabbit meat producer (Li et al., 2018). Up to 2021, meat market has continued expanding modestly, with China followed by North Corea and Egypt as the main consuming countries (IndexBox, 2021). Cuniculture has a key role on diversification of the meat production sector in an increasingly demanding society from the food point of view. Accordingly, new farming strategies as well as national and international regulations on animal welfare, organic farming and climate change are urgently needed to ensure the sustainability of the sector, specifically in the EU (Cullere and Zotte, 2018). In this PhD thesis, we are setting the baseline for the application of pheromones in cuniculture as a way of reducing the GENERAL INTRODUCTION 101 use of injectable hormones, thus creating a more ‘natural image’ for the sector. In a broader scenario, pheromones might contribute to enhance animal production and welfare, covering both producers’ and consumers’ expectations. 4.4.2 Reproductive performance in rabbit farms Since the end of the last century, the artificial insemination (AI) has been established as the most commonly used reproductive system in rabbit farms. Female rabbits are induced ovulating animals and lack of a defined and regular estrous cycle (Arias-Álvarez et al., 2010). Therefore, to perform AI animals need to be synchronized in their maximum receptivity stage. Currently, this is induced by hormone treatments of GnRH and eCG (equine chorionic gonadotropin). Regarding GnRH, advances have been made and nowadays this hormone is included in the insemination straw, thus reducing animal handling (Vega et al., 2012). As for eCG, despite it can be administrated in several consecutive cycles without having unwanted effects (Maerten et al., 1995), it implies animal handling and greater workforce. Additionally, despite hormones are applied to mothers and not enter in the food chain, there has been an important discussion about the possibility of replacing hormones with different bio-stimulation methods for preserving the ‘natural image’ of rabbit meat. 4.4.3 Biostimulation methods Biostimulation is an animal management practice which helps improving reproductive efficiency by modulating animal’s interaction with external sensory stimuli (visual, olfactory, pheromone, tactile, auditory, social and nutritional cues, among others). In most cases it allows individuals to develop their own natural behaviour, also contributing to the improvement of animal welfare (Theau-Clément et al., 1998). In rabbit farms, different biostimulation methods are usually employed –mostly in conjunction– to improve animal production: mother-litter separation (Garcia‐Dalmán and González‐Mariscal, 2012), feeding control PAULA RODRÍGUEZ VILLAMAYOR 102 (Quintela et al., 2001), photoperiod programs (Mousa-Balabel, 2011), animal manipulation and, the called ‘male effect’ (Theau- Clement, 2008). Additionally, various biostimulation methods are generally used in conjunction with hormone treatment in doe rabbit farms to ensure reproductive efficiency (Keeling et al., 2019). 4.4.3.1 Biostimulation methods based on pheromone communication The most powerful method of biostimulation is probably that which uses chemical or pheromone communication as a source of sensory stimuli. In fact, the terms ‘pheromone communication’ and ‘biostimulation’ have been wrongly interchanged by the literature (Rekwot et al., 2001; Kerketa et al., 2017). We should then clarify that pheromone communication is used as one type of biostimulation, and relies on chemosensory cues to modulate individual behaviour, usually to enhance their reproductive performance. One example of biostimulation method based on pheromone communication –probably in conjunction with other visual and auditory cues– is the commonly called ‘male effect’, in which females exposed to sexually active males trigger activation of luteinizing hormone (LH) secretion and synchronized ovulation (Gelez and Fabre-Nys, 2004). This practice is a valuable management tool exploited in small ruminant (Walkden-Brown et al., 1999; Martin et al., 2004; Gelez and Fabre-Nys, 2004) and swine (Chenoweth et al., 2014) husbandry to stimulate the onset of puberty and to reduce the postpartum period. In rabbits, there is evidence that the presence of males increases the receptivity of females (Lefêvre et al., 1976), its fertility (Berepudo et al., 1993) and also induces sexual maturity in prepubertal rabbits (Frank, 1966). More recent studies point to an improvement of doe reproductive performance (El-Azzazi et al., 2017), especially in does at first lactation (Bonnano et al., 2003) when they are exposed to male odors just before AI. However, published data are controversial (Kustos et al., 2000; Ola et al., 2012), hampering clear conclusions. Additionally, rabbit farms have implemented a biostimulation method based in female-female GENERAL INTRODUCTION 103 interaction prior to AI, which seems to increase their receptivity, but their actual efficiency in terms of fertility and prolificity remain to be elucidated. All in all, pheromone communication has been largely approached from various independent disciplines but without an integrative approach between them, which would definitely offer a deeper understanding not only of the knowledge of chemical communication but also regarding pheromone applications in the industrial sector. For this reason, this work has involved a multidisciplinary approach, involving anatomy, genetics reproduction and behaviour. 111 MATERIAL AND METHODS (In brief) We provide here a brief description of the material and methods employed in this work. More detailed information can be found in the material and methods section of each chapter. All animals pertained to a commercial hybrid -Hyplus strains PS19 and PS40 for female and male, respectively- and were maintained on a farm (Cogal SL, Rodeiro, Spain) under the same temperature conditions (18-24 °C), dark-light cycles of 12:12 hours and ad libitum feeding and drinking. All individuals were humanely sacrificed by an abattoir of the same company, in accordance with the current legislation. 1. ANATOMICAL STUDY 1.1 Animals - Vomeronasal organ: 20 individuals (males and females) of 60- 70 days old. - Accessory olfactory bulb: 10 individuals (males and females) of 3-10 months old. Additionally, 12 individuals (6 males and 6 females) of 70 days old for morphometric studies. 1.2 Techniques - Dissection and microdissection: Identification and extraction of the VNO and the olfactory bulb (OB), exposing the innervation from the VNO to the OB. Aspects such as the PAULA RODRÍGUEZ VILLAMAYOR 112 irrigation and innervation of the VNO and its communication with the environment were studied, determining whether this is done directly through the nasal cavity or indirectly and doubly through both nasal and oral cavities. - Histological processing of the samples, included in paraffin for cutting into microtome (5-10 μm thickness). Both routine (Hematoxylin-eosin / Nissl stain) and specific histological stains (PAS, Alcian Blue, Gallego’s Trichrome, Tolivia and Bielschowsky) were used. - Immunohistochemical and histochemical techniques to obtain morphofunctional information. Lectins such as LEA, BSI-B4 and UEA and antibodies against proteins such as Gαo and Gαi2, OMP, GFAP, GAP-43, MAP-2, glutaminase and calcium binding proteins (CB, CR) allowed us not only to discriminate the different cellular components of both formations (VNO and OB), but also to assess the expression of the two most important subfamilies of vomeronasal receptor, - V1R and V2R- known to be associated to Gαi2 and Gαo expression, respectively. - Morphometric and stereological analysis of the rabbit AOB in both males and females to study sexual dimorphism. 2. GENOMIC STUDY 2.1 Animals 24 animals for studying the rabbit VNO transcriptome, evaluating differential gene expression between different conditions, and assessing VNO plasticity. There were 3 animals per experimental condition, as follows: juvenile (40 days) and adult (180 days) males and females, and two different environmental scenarios: sex-separated and sex-combined. 2.2 Technique We employed RNAseq. After sacrificing the animals, swift dissection of the VNO was MATERIAL AND METHODS (IN BRIEF) 113 needed to avoid RNA degradation. The double VNO structure was immediately dissected out after opening the lateral walls of the nasal cavity and removing the palate and nasal turbinates. Samples were immersed in Trizol and kept in ice (~4°C). Tissue was homogenized at the sampling point using a mixer to guarantee the whole tissue sample is soaked by Trizol -due to the double bone and cartilage envelope of the rabbit VNO-. After 20 minutes, samples were stored at -80°C for further RNA extraction (RNeasy mini kit (Qiagen) with DNase treatment). RNA was sent to Novogene UK Company SL (Cambridge) for library construction and sequencing. The bioinformatics analysis included quality filtering (FastQC) and elimination of adapters and barcodes (Trimmomatic v3.0). Filtered reads were aligned against the rabbit genome (OryCun2.0) and assigned to genes based on the latest annotation of the rabbit genome using STAR v.2.7.0e two-pass mode. Additionally, Kallisto, a software that pseudoaligns reads to a reference genome producing a list of transcripts that are compatible with each read while avoiding alignment of individual bases, was also used to compare the results between STAR and Kallisto. 3. FIELD STUDY 3.1 Animals The behavioural study was done in groups of 60 females. According to the experimental design, there were five experimental groups and the experiment was repeated three times over a 120-day period (once each 40 days, according to the female reproductive cycle). Even though some individuals were the same at different experimental time-points, we consider them as ‘different individuals’ because the data obtained was new each time. In total, for the behavioural study we employed 60 x 5 x 3 = 900 females in reproductive stage. Urine was obtained from 25 mature males and 25 mature females (> 180 days) by ultrasound-guided cystocentesis 24 h before the behavioural experiment (and kept at 4 ºC overnight). Considering that PAULA RODRÍGUEZ VILLAMAYOR 114 the experiment was repeated three times, we employed a total of 75 males and 75 females. Seminal plasma was provided by an AI center from 60 mature males (>180 days) 24 h prior to the behavioral experiment. Ejaculates were mixed together and centrifuged at 3000 rpm, 10 min, to obtain the seminal plasma, which were then kept at 4 ºC overnight. Similarly, 60 x 3 experimental time-points = 180 mature males. 3.2 Experimental design Evaluation of specific female rabbit (doe) reproductive parameters when they are exposed to biological fluids –urine and seminal plasma of adult individuals (6 months old)–. Groups of 60 females/each (between third and seventh birth) were exposed to (1) female urine, (2) male urine, (3) seminal plasma and (4) female– female (F–F) separated, just before AI, and compared to a ‘golden method’ F-F interaction. The following reproductive parameters were analyzed for each group: receptivity (vulvar color), fertility (kindling rate), prolificacy and number of born alive and dead kits/litter. For conditions 1, 2 and 3, the corresponding stimulant was sprayed around the nose area, 1 h, 15 min, and 1 min before insemination. Specifically, 1 mL nasal spray was used in each exposure per animal, in total, 3 mL / individual. AI and the corresponding handling were always performed by the same farm workers to reduce statistical noise. CHAPTER I Morphological and immunohistochemical study of the rabbit vomeronasal organ Paula R Villamayor, José Manuel Cifuentes, Patricia Fernández de Troconiz, Pablo Sánchez-Quinteiro* (2018) Morphological and immunohistochemical study of the rabbit vomeronasal organ. Journal of Anatomy 233:814—827. doi: 10.1111/joa.12884. Department of Anatomy, Animal Production and Clinical Veterinary Sciences, Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain. *Corresponding author. CHAPTER II Structural, morphometric and immunohistochemical study of the rabbit accessory olfactory bulb Paula R Villamayor1, José Manuel Cifuentes1, Luis Quintela2, Ramiro Barcia3, Pablo Sánchez-Quinteiro1*(2020) Structural, morphometric and immunohistochemical study of the rabbit accessory olfactory bulb. Brain Structure and Function 225:203–222. doi: 10.1007/s00429-019-01997-4. 1Department of Anatomy, Animal Production and Clinical Veterinary Sciences, Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain. 2Department of Animal Pathology, Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain; 3 Department of Biochemistry and Molecular Biology, Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain. *Corresponding author. CHAPTER III Analysis of the vomeronasal organ transcriptome reveals variable gene expression depending on age and function in rabbits Paula R Villamayor1,2, Diego Robledo3, Carlos Fernández1, Julián Gullón4, Luis Quintela5, Pablo Sánchez-Quinteiro2*, Paulino Martínez1 (2021) Analysis of the vomeronasal organ transcriptome reveals variable gene expression depending on age and function in rabbits. Genomics 113:2240–2252. doi: https://doi.org/10.1016/j.ygeno.2021.05.007. (Open Access). 1Department of Zoology Genetics and Physical Anthropology, Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain; 2Department of Anatomy, Animal Production and Clinical Veterinary Sciences, Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain; 3The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, UK; 4Conejos Gallegos, COGAL SL, Rodeiro, Pontevedra, Spain; 5Department of Animal Pathology, Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain. *Corresponding author. 309 DISCUSSION This discussion highlights the importance of approaching pheromone communication from an integrative perspective, considering both basic research (i. e. structural, molecular, behavioural approaches, etc.) and applied research to the industrial sector. This means that pheromone market should always go hand with hand with research studies to ensure final product efficiency and security. Additionally, due to their fundamental role in pheromone perception, vomeronasal receptors –VRs, FPRs and sex-steroid receptors– are also extensively approached in this discussion. We will pay special attention to the plastic capacity of sex-specific VRs upon environmental modulation and discuss their potential implication in the ‘male effect’. We will also highlight the extranasal VRs gene expression and their potential functional implications. Finally, we will point towards new vomeronasal receptors yet to be discovered and provide evidence of sex-steroid receptors as possible pheromone receptor candidates. Overall, the high complexity of pheromone perception and in particular of vomeronasal receptors, calls for further investigations into their physiological and molecular rationale. 1. IMPORTANCE OF AN INTEGRATIVE APPROACH IN CHEMOCOMMUNICATION RESEARCH As we stated throughout this manuscript, pheromone-mediated chemical communication plays a fundamental role in animal reproduction and physiology. Our main goal was to gain an in-depth understanding of chemocommunication mediated by the VNS in rabbits through a multidisciplinary approach that combines traditional morphofunctional analysis (chapter I and II) with innovative PAULA RODRÍGUEZ VILLAMAYOR transcriptomic and molecular methodologies (chapter III and IV), but also, behavioural field studies that measure reproductive parameters related to pheromone exposure (chapter V). A main outcome of this approach would be transferring results to the productive system by applying pheromonal compounds for improving rabbit farming, ameliorating reproductive parameters and animal welfare, as well as reducing the usage of hormones in farms. Getting into the detail, the structural and morphofunctional study of the rabbit VNO (chapter I) shows a well-developed structure, with an extensive venous vasculature, which differs from any other group of mammals (Vaccarezza et al., 1981; Barrios et al., 2014). This, along with a double bone and cartilaginous envelope, strongly suggests that the rabbit VNO is very active at receiving pheromones by pumping VNO mechanisms (see discussion of chapter I for detail). Also, the expression of the two vomeronasal G-protein families –gαi2 and Gαo– in the rabbit VNO neuroepithelium (chapter I) and in two segregated regions of the AOB –Gαi2 at the anterior part and Gαo at the posterior part (chapter II)–, likely correlates to the expression of VRs, since a link between the expression of V1Rs with Gαi2 and V2Rs with Gαo is generally assumed–. Remarkably, preliminary data from our group have shown that in rabbits at perinatal stage there is no correlation between the expression of VRs and their corresponding G- protein (Villamayor et al., 2019; 2022a). This is rather surprising and contradicts most of the VNO traditional studies to date (Halpern and Martinez-Marcos, 2003; Suarez et al., 2011a). Notwithstanding, it demonstrates the importance of approaching VNS studies from an integrative perspective (morphofucntional vs gene expression studies in this case) and points towards the necessity of more in-depth studies regarding the real association between V1R and V2R with Gαi2 and Gαo, respectively. The broad diversity of VRs together with the lack of specific antibodies hampers their mophofunctional characterization. Genomic data complement very well structural studies. We employed gene expression analysis (RNAseq) to identify and update the active VR repertoire in rabbits –we found 128 V1Rs and 67 V2Rs– (chapter III). The number of VRs greatly varies among species (from > 250 in mice DISCUSSION to ~ 8 in dogs) (Grus et al., 2005; Young and Trask, 2007), and comparisons of VR repertoires and their gene expression patterns have been done across a range of mammals, demonstrating that each species has a ‘quasi-private’ repertoire to detect and respond to a wide variety of external signals (Wynn et al., 2012). We also analyzed the rabbit VNO gene expression variation across different socio-environmental conditions (chapter IV). Indeed, we identified that VRs were especially up- and downregulated in sex-separated juvenile females and sex-separated adult females, respectively. Our data revealed that VRs expression repertoire in rabbits is environmentally modulated which contrasts with results obtained from a similar experimental study in adult mice (Van der Linden et al., 2018) (see discussion of chapter IV for details). If we translated our rabbit data into a rabbit farm perspective, where males and female are usually located in separated facilities, we would expect that females not exposed to males –rabbit farm routine– would display down-regulation of their VRs repertoire –according to our sexseparation study (chapter IV)–, and this could be linked to a lower reproductive performance. Then, if we were to expose females to sexually mature males –the so-called ‘male effect’, which is known to increase female receptivity and fertility (Gelez and Fabre-Nys, 2004; El-Azzazi et al., 2017)–, how would VRs expression behave? In other words, are VRs implicated in the ‘male effect’ and if so, how would their gene expression patterns be like? The physiological rationale underlying ‘the male effect’ remains largely unknown, but pheromones are likely to be the main key players at driving such behaviour. Therefore, we would expect a direct functional implication of the VNO and VRs in the ‘male effect’. Knowing that adult females show overall down-regulation of VRs when sex-separated (chapter IV) and assuming, that exposure to males (‘male effect’) would lead to an increase in reproductive performance, the ‘male effect’ could be guided by an increase of VRs expression in females when exposed to sexually active males. Importantly, since the VNO has shown experience dependent plasticity (Marom et al., 2019), this would likely explain why the ‘male effect’ is ‘acute’ –meaning that it is effective only if the exposure takes place during a short PAULA RODRÍGUEZ VILLAMAYOR period of time–. Instead, chronic exposure to males eventually leads to a drop in female reproductive performance (Gelez and Fabre-Nys, 2004). This fact could be explained by the saturation of VRs and the consequent reduction of their expression (see theory use-it-and-lose-it in discussion of chapter IV). Further behavioural and gene expression analysis are needed to verify this hypothesis. Since we have already established a protocol for testing female reproductive performance following ‘pheromone exposure’ via biological fluids (chapter V), and we also have long experience with gene expression analyses (chapter III, IV), we are equiped with the experience to design experiments aimed at testing the functional implication of VRs in ‘male effect’. At a more basic scientific level yet essential to know how the VNS drives behaviour, a further step would be to identify how those vomeronasal inputs are processed in the brain. We have provided an extensive structural and morphofunctional analysis of the rabbit AOB, but also determined its structural sexual dimorphism -female rabbit AOB presents higher morphometric values than male (chapter II)-. This is in accordance with previous data from Guillamón and Segovia (1997), who also identified sexual dimorphism in rats, but with higher morphometric values in male than in female. We have no explanation for these opposite patterns between the two species, and the functional role of such AOB dimorphism remains unknown. In any case, we argue that the structural AOB sexual dimorphism may play a role at mediating sex-specific behaviours. The nature of the AOB is highly complex due to continuous neuroregeneration of inhibitory interneurons that somehow regulate the signaling of principal cells. Considering the importance of these newborn neurons in the regulation of reproductive and social behaviour, further studies in rabbits should aim at deciphering their functional implication in the AOB circuit, and it might be wise to address the studies in a sexspecific manner. In order to do so, we could take advantage of a recent study that has provided insights into the complex physiology of mice AOB interneurons, and which set the basis for future studies of AOB function, circuits and plasticity mechanisms (Maksimova et al., 2019). Also, new OMICs techniques, such as single-cell RNAseq and spatial transcriptomics, are providing new insights into the cellular DISCUSSION heterogeneity of adult born MOB interneurons as well as the complex organization of the MOB glomerular map in mice (Tepe et al., 2018; Wang et al., 2022). These cutting-edge techniques have not yet been applied to the AOB, but they constitute promising opportunities to tackle the molecular rationale of the AOB. Therefore, multidisciplinary approaches including new –OMICs but also new imaging techniques, as well as electrophysiological recordings would be crucial to understand the functional and structural interplay of this tinny structure, extremely necessary for animal reproduction and survival. All in all, pheromone communication has been largely approached from various independent disciplines, but without an integrative strategy, which would definitely provide a deeper understanding, not only of the knowledge of chemical communication, but also regarding pheromone applications for the industrial sector. For this reason, this doctoral thesis approaches the VNS from anatomical, genomic, behavioural and reproductive points of view, and other new approaches such as proteomics and volatolomics are already in the track. As a result, it is expected an improvement in the reproductive parameters and well-being of farmed rabbits through pheromone implementation. 2. COMPLEXITY OF VOMERONASAL CHEMORECEPTORS Vomeronasal receptors have evolved independently in the different taxa and species to detect a broad range of chemical stimuli and instruct specie-specific behaviours. Despite many advances in the field have been made in the past few years (reviewed in Tirindelli, 2021), we still do not know how pheromones / chemical cues bind vomeronasal receptors, and therefore approaching the study of these receptors in different species and from a multidisciplinary perspective will help framing how this system works and ultimately how it affects species-specific behaviours. This part of the discussion exposes the high complexity of vomeronasal receptors, with special focus on the VRs gene expression variation depending on socio-environment conditions as well as its expression in extranasal tissues. Also, due to PAULA RODRÍGUEZ VILLAMAYOR the identification of a new family of vomeronasal receptors –FPRs– which was exclusively found in mice (Liberles et al., 2009; Rivière et al., 2009), we raise the question of whether there may be speciesspecific families of vomeronasal chemoreceptors yet to be discovered. Finally, the expression of sex-steroid receptors in VSNs together with the fact that steroids act as VNO stimuli are rather new concepts which open new avenues for studying whether such sex-steroid receptors could be identified as a new type of vomeronasal receptors. 2.1 V1R and V2R receptors V1R and V2R receptors are among the gene families showing broader variation in gene number across vertebrate lineages (Nei et al., 2008; Shi and Zhang, 2007). Due to this species-specificity, vomeronasal studies should always be addressed in each species independently and from a comparative point of view among species. Extrapolations from one species to another might lead in most cases to mistaken conclusions (Salazar and Sanchez-Quinteiro, 2009). Additionally, we should keep in mind intraspecific variation as a source for local adaptation of populations and further as the raw material for evolutionary studies. We performed a phylogenetic study of the rabbit VRs and compared it to that of mice –a phylogetically close species– (chapter III). Our data revealed that rabbits have undergone several expansions from ancestral genes, which have been completely lost in mice, and that are distributed in a few independent clades. Additionally, VRs seemed to group in clusters in the rabbit genome. For example, we found 30 V1R genes in chromosome 9. However, most of the VRs appeared in scaffolds not anchored to chromosomes, and although they may also be grouped, a new rabbit genome assembly will be necessary to appropriately address this question. In contrast to mice, in which VRs have been extensively classified in the literature (Yang et al., 2005; Miller et al., 2020), rabbit VRs have not yet been grouped in different subfamilies. In an attempt to classify them, we contacted Prof. Leif Anderson´s group from Uppsala University, which recently released a new rabbit genome assembly, OryCun 3.0 (2020), but this DISCUSSION genome is not annotated and therefore we could not use it in our analysis. According to Prof. Anderson, this assembly would not be annotated due to specific issues related to old PacBio chemistry, which produced small gaps in the assembly, but a new OryCun 4.0 assembly is in the way, which will be critical for future VNO studies, especially considering the evolutionary history of vomeronasal receptors. It is well documented that VNO-mediated behaviour is sexually dimorphic (i. e. male pheromones such as MUPs or ESP1 evoke territorial aggression when detected by another male, but promote sexual attraction and receptivity when detected by females) (Tan and Stowers, 2020). Morphological sexual dimorphism has been found at every stage of the vomeronasal pathway, including the VNO, AOB, amygdala, and hypothalamic areas (Guillamón and Segovia, 1997; Segovia et al., 2006; chapter II). However, the first evidence of functional sexual dimorphism occurs in the MeA (Bergan et al., 2014; Tan and Stowers, 2020), thus suggesting that both sexes may equally detect cues, which in turn will be differentially processed in each sex at higher brain centers. This hypothesis is also supported by the fact that VRs expression was found to be similar in male and female mice exposed to the same environmental conditions (Ibarra-Soria et al., 2014b). Consistent with this, our data demonstrated that, in rabbits, VNO and VRs expression patterns follow a similar logic in males and females exposed to the same environmental scenario (chapter III). Nevertheless, considering that the VNO is a sensory organ which takes over sensing a broad range of chemical stimuli from the external world, this landscape –meaning the similar VNO and VRs expression patterns found in males and females exposed to similar environments– could change if animals are subjected to different environmental conditions. Indeed, studies in mice showed that the VNO is able to adapt responses to a given scenario, demonstrating its experience- and statedependent plasticity (Lanuza et al., 2014; Mohrhardt et al., 2018; Marom et al., 2019; Trouillet et al., 2021; Villafranca-Faus et al., 2021). Also, VRs gene expression has proven to be affected sexspecifically by particular environments in mice (Van der Linden et al., 2018). In our study, we showed for the first time the rabbit VNO PAULA RODRÍGUEZ VILLAMAYOR plasticity under different socio-environmental conditions (chapter IV). Specifically, our sex-separation vs sex-combined experimental approach, where animals do not contact with members of the opposite sex since birth vs in close contact since birth, revealed that VNO and VRs gene expression differs between males and females under the two studied environmental conditions. This result proved that despite sex itself does not influence VRs expression as long as animals are exposed to same environment (demonstrated in rabbits in chapter III), such expression dramatically changes when animals are exposed to different scenarios (chapter IV). We argue that sexual dimorphism of the VNS and specifically of the VRs should always be considered according to the environment to which animals are exposed to, because despite sex might not be a condition itself, responses to a given environmental condition are definitely shaped in a sex-specific manner. Additionally, considering that the VNO has been suggested to play a role at puberty onset (Szymanski and Keller, 2014; Cross et al., 2021), we extended our study to juvenile individuals. When comparing the gene expression of juvenile rabbit VNO to that of adults –under same environmental conditions–, striking differences were detected between the two stages. However, the VR repertoire showed little gene expression differences between the two stages (chapter III). This would follow a similar logic as the one described above for male and female adults under the same environmental conditions –equal capacity for detecting cues through the VNO, but different ways of processing the information at higher brain centers–. Our next question was to determine whether juvenile VNO and VRs expression would also be affected by exposure to a particular environmental, and if so, whether this would also be sex-specific. Following a similar approach as outlined above, we determined that juvenile VNO and VRs gene expression repertoires sharply vary upon environmental conditions in females but not in males, thus pointing to a functional role of the VNO at the onset of puberty (see discussion of chapter IV for details). Finally, the DEGs repertoires found between sex-separated and sex-combined conditions greatly differ between juveniles and adults, thus proving that the VNO and VRs not only DISCUSSION show sexbut also stage-specific gene expression, and this is conditioned by a given environment. All in all, considering that until now chemical cues / pheromones were thought to be equally detected in males and females by the VNO and VRs, our data strongly suggest that the VNO and VRs are highly sensitive chemodectors with an outstanding plasticity to adapt to a continuous changing environment, and this is sex- and stage-specific. 2.1.1 Extranasal expression of VRs An additional level of complexity of vomeronasal receptors comes from their extranasal expression. Outside olfactory organs, V1Rs expression was found in testis in mice and swine (Tatsura et al., 2001; Dinka et al., 2016), but also in brain, bulb, heart, kidney, and liver (< 7 genes / tissues) (Zhang et al., 2010), though their function remains unknown. As for V2Rs, despite the vast majority of V2R genes were only expressed in the VNO, Ibarra-Soria et al., (2014) found expression of one V2R in the MOE, Vmn2r29, suggesting previous unrecognized mechanisms of pheromone detection in the MOE. In other non-sensory tissues, only very few V2Rs were found, specifically in mice brain, bulb, heart, kidney, liver, and lung (< 5 V2Rs / tissue) (Zhang et al., 2010), but as for V1R, their potential functionality remains completely unexplored. In rabbits, we compared the VRs VNO expression to that of other seven rabbit tissues (hindbrain, forebrain, ovary, testis, liver, heart and kidney) available in the Rabbit Expression Atlas (Cardoso-Moreira et al., 2019). We found high expression of extranasal VRs in testis, low expression in ovary and brain and no expression was found in heart, liver and kidney (see chapter III for detail). Despite VRs extranasal expression has not been comprehensively addressed in the literature, studies of extranasal ORs expression point to the necessity of further approaching this topic. Briefly, the traditional definition from Buck and Axel (1991) stated that ORs were only found in the main olfactory epithelium. However, a small subset of ORs was later found expressed in other chemosensory tissues such as the VNO (Lévai et al., 2006) and the septal organ (Kaluza et al., 2004; Tian and Ma, 2004). Additionally, soon after the discovery of ORs, their expression was PAULA RODRÍGUEZ VILLAMAYOR the foundation to continue this work into the final goal: pheromone commercialization. Up to date, a few ‘putative pheromones’ have been commercialized, especially for dogs and cats (to control behaviour) but also for some farmed species (to control reproduction). However, most of them lacked scientific background, leading to inefficient products. For example, in rabbits the Rabbit Mammary Pheromone – 2MB2– is known to exclusively induce pup suckling behaviour (Coureaud et al., 2010; Charra et al., 2013; Schneider et al., 2016). Industrial market has taken advantage of this only characterized rabbit pheromone to date -2MB2-, by commercializing an analog called Rabbit Appeasing Pheromone, described as ‘pheromone products based on a combination of pheromones’ including the rabbit mammary pheromone 2MB2 (i.e. 1); Ceva Santé Animale created the first ‘Rabbit Appeasing Pheromone’ (http://www.asfclapin.com/Docs/Activite/cuninov/Cuninov2007/Cuninov2007- CEVA.pdf, 2007 3 ; 2); and Sibpma and SIGNS laboratories have commercialized the SecureRabbit®, a synthetic analog of the maternal appeasing pheromone (licence IRSEA – US Patent 6-077-867, 6-054- 481 y 6-169-113) (https://sibpma.es/wpcontent/uploads/2020/12/Ficha-SecureRabbit%C2%AE-SIGNS- 2020.pdf) 5 . However, there are no published data (neither VNS neuroanatomical studies (sensory pathways involved, receptor IdeaLugo prize: https://www.elprogreso.es/gl/articulo/lugo/proxecto-mellora-benestar-animal- feromonas-fai-premio-idealugo/202011251340161471891.html 3 CEVA. According to their results, female rabbits were less stressed, and technical actions were easier. Moreover, fertility (percentage of parturitions per artificial insemination (AI), live born rabbits per litter and rabbits’ viability at birth improved (Bouvier et al., 2008, conference communication). This product is not available in the CEVA website, suggesting that is not being commercialized – we have no information whether it has ever been commercialized. 4 SIBPMA pheromones prevent the negative effects of stress, improve animal production and enhance animal welfare. However, no scientific data has been reported and its efficiency should be considered with caution. 5 SIBPMA pheromones prevent the negative effects of stress, improve animal production and enhance animal welfare. However, no scientific data has been reported and its efficiency should be considered with caution. DISCUSSION locations, etc.) nor any functional approach (gene expression, electrophysiology, neuroendocrine studies, etc.)) that justify the use of a ‘newborn suckling pheromone’ to improve the well-being of adult rabbits. In this context, it is important to highlight that pheromone market is not regulated by law. Indeed, a direct consult to the Spanish Agency of Medicines and Medical Devices (AEMPS) in 2021 indicated that since pheromone market is quite small and lacks specific legislation, this type of compounds could be considered under different laws depending on their characterization / identification processes (i. e. in Spain, ‘Law 29/2006’ if we consider pheromones as compounds obtained from glands). Some companies have taken advantage of this gap to sell ‘pheromone products’ without any scientific background, which –not surprisingly– turn out being extremely inefficient. It is therefore of utmost importance that the scientific community within the field tackles this issue, asking for the implementation of specific regulations in which pheromone products must prove a solid background which justifies its effectiveness before being sold. CONCLUDING REMARKS AND FURTHER STEPS This doctoral thesis set the basis for chemocommunication research in rabbit. It provides strong foundation of the structural and molecular basis of the VNS and specifically of VRs, as well as establishes a protocol for further field studies on pheromone-induced behaviours. Started from scratch five years ago, this project has already generated a great deal of relevant data which may ultimately contribute towards the competitive implementation of pheromones in the market. This work is included in a broader project of our group. The next steps include 1) molecular studies (proteomics, volatolomics, etc.) of biological fluids to characterize rabbit pheromones; 2) behavioural analysis of biological compounds other than urine and seminal plasma, such as extracts from exocrine glands, to study their impact in reproductive parameters of female and also male rabbits; 3) establishing the link pheromone-receptor by molecular studies (qPCR, in situ hybridization); and 4) field studies with potential pheromone candidates. All these analyses will benefit from the data obtained in this PhD thesis. The final goal of this approach is to improve animal production and welfare by the implementation of pheromones as natural molecules that contribute to the ecological sustainability of livestock production. Indeed, the validity of these results as well as of the coming research approaches have been recently supported by the Spanish Science and Innovation Ministry, with a 3-year project for their continuation. CONCLUSIONS 331 CONCLUSIONS 1. Vomeronasal chemoperception is highly complex and should always be approached from a multidisciplinar perspective. Our data provide extensive knowledge into rabbit chemocommunication, specifically at anatomical, genomic, reproductive and behavioural levels, providing the baseline for further translational studies aimed at implementing the use of pheromones in rabbit farms. 2. At anatomical level, the rabbit, Oryctolagus cuniculus, holds a well-developed VNS –VNO and AOB–, which contains all necessary elements to develop an effective chemocommunication between individuals of the same species. 3. The degree of structural development of the adult rabbit VNO is comparable to that of mammals with the highest level of chemocommunication. The VNO displays many speciesspecific morphological features, such as a double direct and indirect communication –via nasal and oral cavities, respectively– to the external world. Pheromone circulation along the VNO length is ensured by its double bone and cartilaginous enveloped as well as its unique blood vessels. 4. The adult rabbit AOB has a complex structure with specific topographic, lamination and neurochemical properties. It also contains three types of principal cells that significantly differ PAULA RODRÍGUEZ VILLAMAYOR in quantity between males and females, indicating structural sexual dimorphism. Additionally, four neuronal clusters composed of piramidal-like cells and found at the accessory bulbar white matter suggest an additional level of complexity of the AOB circuitry. 5. Immunohistochemical and histochemical markers are valuable tools for characterizing the morphofunctional features of the rabbit VNS, especially the AOB. For instance, G-protein Gαi2 and Gαo allowed the identification of two well-differenciated AOB regions –anterior and posterior, respectively–. 6. At genomic level, the rabbit VNO transcriptome does not differ between males and females, but it significantly differs between adults and juveniles. It contains 128 and 67 V1Rs and V2Rs, respectively, as well as many genes involved in reproduction, immunity and VNO functional activity. 7. The rabbit VNO is highly plastic and shows sex- and stagespecific gene expression differences upon socio-environmental conditions –via sex-separation and sex-combined scenarios-. VRs gene expression is significantly down- and upregulated in sex-separated adult female and sex-separated juvenile female respectively. Similarly, genes involved in reproduction, immunity and functional activity are also highly plastic in terms of their gene expression patterns. 8. Biostimulation methods employing urine and seminal plasma as source of pheromones did not affect the reproductive performance of female rabbits. 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