1. MAJOR HISTOCOMPATIBILITY COMPLEX
The Major Histocompatibility Complex (known as Human Leukocyte Antigen system in humans) is a set of cell-surface proteins which mediate the interaction between body cells and the immune system.
Three classes of MHC proteins are known, the first two being the ones to determine recognition of self or non-self cells: these proteins belong to the immunoglobulin superfamily and possess a groove to which short polypeptides (8 to 20 amino acids) are bound.
Class I MHC proteins are present on virtually every nucleated cell in the organism (thus not eritrocytes), providing recognition of self cells to CD8+ T-lymphocytes, while class II MHC proteins are expressed only by Antigen Presenting Cells, whose main duty is to phagocyte exogenous pathogens in order to present them to CD4+ T-lymphocytes.
Besides these immunological functions, in many species such as mice, rats, primates and man MHC proteins seem to have certain importance in mate selection as well: in fact, following experiments by Yamazaki et al. (Control of mating preferences in mice by genes in the Major Histocompatibility Complex, 1976) demonstrating MHC-dependant choice of sexual partner in mice in the late 70’s, Swiss researcher Claus Wedekind conducted during 1995 a brilliant (and cheap as well) experiment, known as the “sweaty T-shirt experiment” (MHC-dependent mate preferences in humans, 1995) in wich 44 women, 18 of whom users of oral contraceptives, were asked to smell and rate T-shirts worn by 49 men for 2 days: the analysis of the preferences showed that women not using contraceptives tended to choose T-shirts belonging to men with dissimilar HLA alleles.
The hypothesis made to explain this preference is that, as well as for other animals in which MHC-dependent mate choice has been demonstrated, offsprings born from such sexual partner would have two main evolutive advantages: the choice of non-self and non-parental MHC-partners determines heterozygosis at the MHC loci and inbreeding avoidance. While inbreeding avoidance reduces the chances of progeny to be homologous for recessive deleterious alleles, heterozygosis for MHC loci delivers better immune protection: since expression of MHC alleles is co-dominant, heterozygotes for these alleles express twice the amount of functional MHC proteins than homozygotes.
Instead, the reason why contraceptive-user woman tend to prefer MHC-similar scents is still not known: hypothesis were made (MHC-correlated odour preferences in humans and the use of oral contraceptives, 2008 and Does the contraceptive pill alter mate choice in humans?, 2009) suggesting that pregnancy, or in this case an hormone-simulated one, draws women towards "nurturing relatives".
It is also known that woman preferences vary during the menstrual cycle, suggesting that hormones may modulate the perception of these olfactive stimuli.
Subsequent experiments (The human brain is a detector of chemosensorily transmitted HLA-class I-similarity in same and opposite sex relations, 2006) used electroencephalography to determine brain responses in both men and women to HLA-similar and dissimilar olfactive stimuli. Results showed that these stimuli activate mainly the frontal cortex in men while the parietal cortex in women showed greater response. Both males and females, though, show bigger and faster responses to HLA-similar stimuli than to HLA-dissimilar ones: the authors’ conclusions suggest that avoiding mating with similar HLA alleles and thus avoiding chances of inbreeding is much more important to the organism than finding the best partner among a group of dissimilar ones, whose odor elicits a weaker response.
Furthermore, these experiments and others show high correlation between MHC dissimilarities and odor preferences when a female smells a male’s scent while this correlation is much weaker when roles are inverted: this probably reflects the need for the female to choose a good partner for her offspring, given that bearing pregnancy and child raising are not male’s main duties in most mammalians. Females seem to look for quality, males for quantity.
The means by which MHC determines odortypes in urine (mice) and axillary excretion (human) is still not clear. Studies by Yamazaki and colleagues involving H-2 congenic mice (where H-2 stands for murine major histocompatibility complex) in a two-arms maze where they were administered different olfactive stimuli coming from both urine and blood serum suggest that “a likely mechanism for odor-type specification may be that soluble MHC-gene products themselves bind circulating odorants selectively, presumably after they have lost their bound peptide, and then release them mainly during the course of renal processing and excretion.” (Odortypes: their origin and composition, 1999)
It is currently believed that proteins bound to the peptide-binding groove of the MHC may produce the odorant. Each MHC protein binds to a specific peptide sequence, yielding a set of uniquely bound peptide-MHC complexes for each individual. During cellular turnover, the MHC-peptide complex is shed from the cell surface and the fragments are dispersed in bodily fluids such as blood serum, saliva, and urine. Recently, it has been shown that receptors in the vomeronasal organ of mice are activated by peptides having similar characteristics to MHC proteins. (Mate choice decisions of stickleback females predictably modified by MHC peptide ligands, 2005)
The term ‘‘Pheromone’’ is based on the Greek words φέρω (to transfer) and ὁρμή (to excite). A pheromone is a secreted or excreted chemical factor capable of acting outside the body of the secreting individual to trigger a behavioral response in members of the same species. They are contained in body fluids such as urine, sweat , and genital mucous secretions. These chemicals may have several functions (i.e. alarm pheromones, food trail pheromones, sex pheromones, and many others that affect behavior or physiology) and are broadly divided into two classes:
• releaser pheromones produce short-term behavioral changes and act as attractants or repellents
• primer pheromones produce long-lasting changes in behaviour or development activating the hypothalamic–pituitary–adrenal axis .
Basic unicellular prokaryotes to complex multicellular eukaryotes use pheromones. Various studies point to the possible existence of chemical communication in humans, although the theme is still debated and the molecules involved remain to be clerly identified. It is thought that they may have a role in choice of the sexual partner .
Our knowledge of pheromonal effects mainly concerns rodents, which live mostly in darkness, making chemical communication essential. Studies showed that small airborne molecules are involved in this communication along with nonvolatile substances such as steroids, peptides and proteins. Air-transport enables volatile pheromones to reach the olfactory system; non volatile substances, however, accompanied by volatile substances in solution, can be detected by the vomeronasal organ (VNO). One example is aphrodisin, a protein found in the vaginal secretions of hamsters which triggers reproductive behavior in young males activating the VNO. Likewise, major urinary proteins (MUPs) are proteins emitted in mouse urine which act as an authentic signature: every mouse produces a distinct set of MUPs. Another peptid with this function is ESP1 which is released in male mouse tear fluids and enhances female sexual receptive behavior. Aphrodisin and MUPs are lipocalins, water-soluble proteins equipped with an internal sac in which small hydrophobic molecules can lodge. Apart from their role as pheromone carriers, these proteins also seem to play a signaling role themselves.
Detection of such molecules involves the Vomeronasal organ or Jacobson's organ, an auxiliary olfactory sense organ that is found in many animals; the standard view of pheromone-sensing was based on the assumption that most mammals have two separated olfactory systems with different functional roles: the main olfactory system for recognizing conventional odorant molecules and the vomeronasal system specifically dedicated to detect pheromones .
The animal vomeronasal organ is located in a forward position near the nasopalatine duct. Information to SNC is transmitted via the accessory olfactory bulbs, which lie behind the main olfactory bulbs, towards the amygdala and the anterior hypothalamus, the latter being directly involved in gonadoliberin (GnRH) secretion and thus in sex hormone activity. The VNO presents a tubular structure: its internal duct is closed at the back and communicates through a small aperture which opens onto the nasopalatine ducts,thus connecting the oral cavity to the nose, or directly into the nasal cavities. The VNO lumen is covered by sensory epithelium containing detector neurons, whose receptor proteins are distinct from the olfactory ones and are divided into two families:
• short extracellular chain proteins (V1R), carried by short-dendrite sensory neurons synapsing at the posterior part of the accessory olfactory bulbs
• long extracellular chain protein (V2R), carried by neurons deep in the epithelium synapsing at the anterior part of the accessory olfactory bulbs.
Both V1R andV2R are GCPR. V1R-expressing neurons recognize small substances such as volatile pheromones and sulfated steroids, while all of the stimulants for V2R neurons are peptidic molecules (Chemosensory signals and their receptors in the olfactory neural system,2013). Receptor proteins activate the vomeronasal neurons by opening particular ion channels (trpc2) which initiate cell depolarization and action potential emission: experimental deactivation of the genes involved in trpc2 synthesis totally and specifically abolishes vomeronasal function, making mice unable to distinguish the sex of partners.
V2R presenting neurons also express a gene family involved in the major histocompatibility complex, which is thought to govern individual recognition. The MHC genes expressed in these cells are the H2-mv genes. H2-Mv molecules have been reported to be associated with V2R molecules and have been proposed to participate in pheromone detection, nevertheless their exact function remain unknown . A curious observation is that H2-Mvs are expressed not only in the dendrites but also in axons of vomeronasal neurons; even in this seat their functional meaning has not been explained (Expression of Nonclassical Class I Major Histocompatibility Genes Defines a Tripartite Organization of the Mouse Vomeronasal System,2008).
ESP1 (exocrine gland secretet peptid 1) is an example of a V2R-expressing neurons-recognized pheromone and it is found in male mouse tear fluids . In order to identify ESP1-responsive neuron type, Haga et al. performed double-staining for c-Fos induced by ESP1 and all candidate V2R genes; as a result, ESP1- responsive neurons were only labeled with V2Rp5 (The male mouse pheromone ESP1 enhances female sexual receptive behaviour through a specific vomeronasal receptor, 2010). Sensory neurons from V2Rp5- decicient mice lacked responsiveness to ESP1, and the mice did not show ESP1-mediated enhancement of female lordosis behavior. Thus, activation of V2Rp5 is necessary for ESP1-mediated enhancement of female sexual behavior.
ESP1-V2Rp5 is the only peptide pheromone-receptor pair identified thus far in mammals. It is a 102 aminoacids monomer with a 0-22 N-terminal signal sequence which is not necessary for his c-fos inducing activity . The molecule purified from male mouse secretion is the result of a proteolytic cut and it is named ESP1 (36-98), obviously it is equally effective. NMR analysis showed that ESP1 includes both a flexible region (residues 22–48 and 98-102) and a structural region (residues 50–98), and these two regions do not interact with each other. The structural region is referred to as the core domain. Deletion analysis was performed to define the functional region of ESP1 using the c-Fos induction assay. Two N-terminal deletion mutants, ESP1 (51–99) and ESP1 (52–99), both of which lack residue Asp50, exhibited markedly impaired c-Fos-inducing activity, whereas the other N-terminal deletion mutants bearing Asp50 retained activity. The C-terminal deletion mutant ESP1 (36–94), which lacks residue Cys95, lost activity, whereas the C-terminal deletion mutants bearing Cys95 did not. The conclusion is that the residues Asp50–Cys95 are the minimal functional region. This region approximately coincides with the core domain (Asp50–Leu98) defined by NMR. Therefore, it is clear that the structural integrity of the core domain, but not the unstructured N-terminal region, is necessary for c-Fos-inducing activity of ESP1. ESP1 contains two cysteines (cys63 and cys95) and they are connected through an intramolecular disulfide bridge. None of cysteine mutant (like c63s or c95s) induced c-Fos expression, highlighting the importance of the disulfide bridge to the biological activity of the protein. ESP1 consists of three helices: two alpha helices, H1 (residues 51–61)and H2 (residues 68–78), and one 3-10-helix, H3 (residues 86–90). The N-terminal residue Asp50 of the minimal functional region (Asp50–Cys95) is involved in the interaction between the Nterminus of theH1helix and the loop connecting the H2 and H3 helices, whereas the C-terminal residue Cys95 forms the intramolecular disulfide bridge to bring the H3 helix in close proximity to the H1 and H2 helices. The molecular surface of ESP1 can be divided into three parts which are shown in the figure below .The surface depicted on the left is rich in positive charges, with Arg56 and Arg60 in H1 and Lys84, Arg92, Lys94, and Arg96 in the neighboring region of H3. The surface depicted in the middle has an abundance of negative charges, represented byAsp50 and Glu53 in H1 and Glu67, Asp69, and Asp74 in H2. Compared with the two surfaces, the surface depicted on the right has no such electrostatic properties. Mutational analyses reveals that this residues located on the charged surfaces of ESP1 are essentials for the activation of the vomeronasal receptors. Furthermore intramolecular disulfide-bridge and Asp50 are not crucial for the global helical structure but are required for the maintenance of the local electrostatic surface for receptor binding.
The ESP1 receptor, V2Rp5, contains a long extracellular N-terminal region, which is characteristic of the class C GPCRs. An in vitro pulldown essay demonstrated that direct binding occurs between ESP1 and this extracellular region of V2Rp5. A soluble recombinant protein comprising the extracellular region of V2Rp5 was incubated with ESP1 that had been covalently attached to agarose beads, and the beads were eluted with free ESP1 or ESP4. ESP4 is expected to bind to a different receptor than V2Rp5, despite the closest ESP family member to ESP1. Immunoblot analysis of the pulldown product revealed that the bound V2Rp5 protein could be eluted with free ESP1, but not with ESP4. These results indicate that ESP1 specifically binds to the extracellular region of comprising the extracellular region of V2Rp5. Then soluble V2Rp5 was incubated with ESP1 attached to beads and the beads were eluted with 500 mM NaCl. The interaction between ESP1 and V2Rp5 so is disrupted by high salt, demonstrating that electrostatic charge-charge interactions contribute to the ESP1-V2Rp5 binding.
To predict the receptor binding mode of ESP1, Yoshinaga et al attempted to build a homology model for the functioning of the extracellular region of V2Rp5, based on the structure of mGluR1, a well known member of the class C GPCRs. mGluR1 functions as a dimer, and its glove-like ligand binding domain in each subunit shifts back and forth between the open and closed forms. The binding of glutamate ligands to the open binding clefts leads to the equilibrium shift to the active state, in which one subunit adopts the closed form and the other remains in the open form, for induction of the signal transduction cascade.The prediction is confirmed by a constructed active state model of V2Rp5 revealing an abundance of negative charges in the open cleft , which electrostatically complements the V2Rp5-binding surface of ESP1. Moreover the volume of the V2Rp5 cleft appears to match the molecular size of ESP1only in the activate state and not in the resting one (G-protein-coupled Vomeronasal Receptor Basis for Specific Binding to the Class C Pheromone ESP1 Reveals a Molecular Structure of the mouse Sex Peptide,2013) .
MHC peptides and MUPs also stimulate V2R-expressing neurons . One of the receptors for MHC peptides includes V2r1b, while the receptors for MUP are yet to be investigated .
Human vomeronasal organ develops in utero. Nerve fibers emerge from the developing organ and travel towards the brain, a crucial step in the development of the reproductive system: as of puberty, gonad functioning depends on hormonal secretion by the anterior hypophysis, governed by GnRH-secreting cells in the arcuate nucleus of the hypothalamus.
These GnRH-secreting cells derive from the olfactory placode, from which both the olfactory and vomeronasal organs develop, and migrate along the vomeronasal axons towards the brain. Defective GnRH cell migration induces hypogonadotropic hypogonadism syndrome (deficient LH and FSH pituitary secretion), which is associated with aplasia of the olfactory bulbs, orbitofrontal cortex alteration in the olfactory sulcus and reduction or absence of olfactory sensitivity.
After this initial development, however, the vomeronasal organ regresses, leaving only a few vestiges in adults. The vomeronasal cavities in adults are located near the vomer bone. Compared to other mammals, the general structure shows many signs of regression: epithelial receptor neurons and underlying nerve fibers towards the brain seem to be absent. The vomeronasal organ is thus nonfunctional in adults.
Other decisive arguments back up this conclusion. The genes coding for V1R-type receptor proteins are mostly deactivated by mutation: only five sequences remain in the human genome (whereas mice have more than 180); the same is true for those coding for V2R-type receptor proteins . Moreover, the genes coding for the trpc2 channels, essential to vomeronasal neuron activation, are again pseudogenes unable to give rise to functional ion channels. Finally, on histologic examination the accessory olfactory bulbs, the target of VNO's axons in the species in which it is functional, are found to be absent.
These arguments lead to the conclusion that the vomeronasal function is inoperative in humans (Vomeronasal organ and human pheromones, 2011).
While humans are highly dependent upon visual cues, when in close proximity smell also plays a big role in sociosexual behaviors . The sense of smell is important as an arousal system that recalls significant environmental events and changes. Olfactory signals were demonstrated to induce emotional responses even if an olfactory stimulus is not consciously perceived, presumably because olfactory receptors not only send projections to the neocortex for conscious processing but also to the limbic system for emotional processing. There are two classes of human putative pheromones : vaginal aliphatic acids and axillary steroids:
• Human vaginal secretions contain various short chain (C2–C6) fatty acids, with a variable composition suggesting a possible correlation with hormone levels during the menstrual cycle. Whether or not human vaginal secretions contain sex pheromones (i.e. copulin) influencing male perception and inducing hormonal changes, is still debated.
• three main axillary steroids have been described as human pheromones: androstenone, androstenol and androstadienone (AND). They are released by the apocrine axillary glands and other sweat glands, which start their activity during puberty upon hormonal influence. Localization of steroid hormone receptors in the apocrine sweat glands of the human axilla, 2005 suggested a possible link between steroid hormone activity and induction of pheromone production by investigating the localization of androgen and estrogen receptors in these glands
Different scientific studies have suggested their role as possible pheromones in humans and in general the existence of human pheromones .
The best-known case involves the McClintok effect (Menstrual synchrony and suppression, 1971 and Regulation of ovulation by human pheromones; 1998), that is synchronization of menstrual cycles among women based on unconscious odor cues. It was found that women smelling samples coming from other women caused their menstrual cycles to speed up or slow down depending on the time in the month the odor sample was collected: before, during, or after ovulation. Therefore, there should be two types of pheromone involved: one, produced prior to ovulation, shortens the ovarian cycle while the second, produced just at ovulation, lengthens the cycle.
Evidence of a role for pheromones in the modulation of sociosexual behavior comes from two double blind, placebo-controlled experiments. The first, by Cutler, had 38 male volunteers apply either a male pheromone or control odor and record six different sociosexual behaviors over two weeks. This study found that there is an increase in sexual behavior in the pheromone users compared to the control group (Pheromonal influences on sociosexual behaviour in men,1998) .
The study by McCoy and Pitino was similar to the Cutler study, only females instead of males were subjects. Females treated with female pheromones reported significant increases in many of the behaviors including "sexual intercourse", "sleeping next to a partner", "formal dates", and "petting/affection/kissing". The researchers believed that the pheromones had a positive sexual attractant effect (Pheromonal influences on sociosexual behaviour in young women,2002).
Van Toller et al. (Skin conductance and subjective assessments associated with the odour of androstanone, 1983) provide evidence of the physiological (and not only psychological) effects of pheromone exposure, showing that skin conductance in volunteers exposed to androstenone was higher than that of non-exposed volunteers. In humans androstenone is the claimed male secreted pheromone that attracts a woman, exerting a positive effect on her mood, cognition and heightening sympathetic nervous system arousal. It is hypothesized that this may be a way for a male to detect an ovulating female who would be more willingly to be involved in sexual interaction .
On the other side androstenol is the putative female secreted pheromone to attract men. In a double-blind study by Kirk Smith (Human social attitudes affected by androstenol, 1978) by Kirk-Smith, people wearing trated or untreated surgical masks with androstenol were shown pictures of people, animals and buildings and asked to rate the pictures on attractiveness. Individuals with masks treated with androstenol rated their photographs as being "warmer" and "more friendly".
Putative pheromones activity can be investigated by means of neuroimaging. Savic and Berglund (Smelling of odorous sex hormone-like compounds causes sex-differentiated hypothalamic activations in humans, 2001) showed that inhalation of androstadienone crystals (a testosterone derivative mostly present in men's axillary secretions) by heterosexual women activates the ventroanterior hypothalamus. The same region is activated in heterosexual men by estratetraenol, an estrogen derivative. According to the authors, the effect as seen on PET scan is too rapid to be due to nasal vessel absorption; eliminating a possible systemic route, the observed activation thus suggests an olfactory contribution. Chronic anosmia abolishes the response to estratetraenol, implying an olfactory role in this activation.(Pheromone Signal Transduction in Humans:What Can Be Learned From Olfactory Loss,2009) In homosexual women, the cerebral activation pattern resembles that of heterosexual men and in homosexual men that of heterosexual women, probably due to sexual dimorphism similarities between homosexual males and heterosexual women and vice versa at the anterior hypothalamus.
Savic and Berglund again (Androstenol: a steroid derived odor activates the hypothalamus in women, 2010) have then demonstrated that androstenol can also activate certain regions of the hypothalamus. PET images of 16 heterosexual women were recorded during passive smelling of androstenol, four ordinary odors, and odorless air. Smelling androstenol caused activation of a portion of the hypothalamus, which according to animal data mediates the pheromone triggered mating behavior. Smelling of ordinary odours, on the other hand, engaged only the classical olfactory regions (the piriform cortex, lateral amygdala, anterior insular and anterior cingulate cortex).
2.3 BIOSYNTHETIC PATHWAY
Though the means by which synthesis of steroid pheromones is expleted in humans is still not completely clear, studies conducted on boar testis homogenates revealed that gonad cells could produce androstenol, suggesting a possible crossing between pheromones and androgens' pathway. Subsequent studies (Comparative biosynthetic pathway of androstenol and androgens, 2001) demonstrated that also human testis produce androstenol and its precursor androstadienone, and that this patway shares at least three enzymes involved in the synthesis of androgens; not only, the same enzymes are also responsible for the synthesis of neuroactive steroids.
Androstadienone is the precursor of steroid pheromones: it is first converted into androstenone by the enzyme 5α-reductase and then reduced to androstenol through the reaction catalyzed by 3β-hydroxysteroid dehydrogenase.
These same enzymes, alongside with 3α-hydroxysteroid dehydrogenase and 21β-hydroxilase (or p450c21), are the same to catalyze the first steps in the synthesis of androgens and neurosteroids; not only, a 2006 study (The pheromone androstenol is a neurosteroid positive modulator of GABA receptors, 2006) states that androstenol may share the functions of both a pheromone and a neuroactive steroid: as well as most of the other neurosteroids it seems to exert a positive modulation on GABAergic neurons.
May pheromones, neurosteroids and androgens be linked and interconnected in their functions and regulation? Inhibition of enzymes involved in these pathways has profound physiological and psychological effects (Allopregnanolone, wikipedia: "Anxiety and depression are common side effects of 5α-reductase inhibitors such as finasteride and dutasteride, and they are believed to be caused, in part, by the prevention of the endogenous production of allopregnanolone"), while synthetic neurosteroids (i.e. ganaxolone, alphaxolone) are capable of strong anaesthetic, anxiolytic, sedative and anticonvulsant effects, so that they are sometimes subministred during treatment of epilepsy.
Indeed a connection between sexuality and psychological wellness mediated by these three classes of steroids seems at least possible.
Responses to MHC and pheromones in humans are dependent on both the individual and context, and their effects are more psychological than physiological, although neurovegetative effects have been reported. Such physiological responses anyway are very far from the big neuroendocrine effects that exposition to these chemicals can elicit in other animals. Their role in human behavior remains speculative and controversial. Human sexuality involves such a diversity of psychological, physiological and cognitive processes that susceptibility to chemical messengers seems slight indeed.
• MHC-correlated mate choice in humans: A review, 2009
• Pheromones in sex and reproduction: do they have a role in humans?, 2012
Airaldi Matteo, Grassini Alberto