Pheromone Molecule Research Articles

Synthesis of Selectively gem-Difluorinated Molecules; Chiral gem-Difluorocyclopropanes via Chemo-Enzymatic Reaction and gem-Difluorinated Compounds via Radical Reaction.

The incorporation of fluorine atoms into an organic compound can alter the chemical reactivity or biological activity of the resulting compound due to the strong electron withdrawing nature of the fluorine atom. We have synthesized many original gem-difluorinated compounds and described the results in four sections. The first section describes the synthesis of optically active-gem-difluorocyclopropanes via the chemo-enzymatic reaction; we applied these compounds to liquid crystalline molecules, then further discovered a potent DNA cleavage activity for the gem-difluorocyclopropane derivatives. The second section describes the synthesis of selectively gem-difluorinated compounds via a radical reaction; we synthesized fluorinated analogues of a sex pheromone of the male African sugarcane borer, Eldana saccharina, and used the compounds as proof for investigating the origin of pheromone molecule recognition on the receptor protein. The third involves the synthesis of 2,2-difluorinated-esters by visible light-driven radical addition of 2,2-difluoroacetate with alkenes or alkynes in the presence of an organic pigment. The last section describes the synthesis of gem-difluorinated compounds via the ring-opening of gem-difluorocyclopropanes. We further developed a novel method of synthesizing gem-difluorohomoallylic alcohols via the ring-opening of gem-difluorocyclopropane and aerobic oxidation by photo-irradiation in the presence of an organic pigment. Since gem-difluorinated compounds that were prepared by the present method have two olefinic moieties with a different reactivity at the terminal position, we accomplished the synthesis of four types of gem-difluorinated cyclic alkenols via the ring-closing-metathesis (RCM) reaction.

Sex Pheromone Receptors of Lepidopteran Insects

The sex pheromone receptors (SPRs) of Lepidopteran insects play important roles in chemical communication. In the sex pheromone detection processes, sex pheromone molecule (SPM), SPR, co-receptor (Orco), pheromone binding protein (PBP), sensory neuron membrane protein (SNMP), and pheromone degradation enzyme (PDE) play individual and cooperative roles. Commonly known as butterfly and moth, the Lepidopteran insects are widely distributed throughout the world, most of which are pests. Comprehensive knowledge of the SPRs of Lepidopteran insects would help the development of sex lure technology and the sex communication pathway research. In this review, we summarized SPR/Orco information from 10 families of Lepidopteran insects from corresponding studies. According to the research progress in the literature, we speculated the evolution of SPRs/Orcos and phylogenetically analyzed the Lepidopteran SPRs and Orcos with the neighbor-joining tree and further concluded the relationship between the cluster of SPRs and their ligands; we analyzed the predicted structural features of SPRs and gave our prediction results of SPRs and Orcos with Consensus Constrained TOPology Prediction (CCTOP) and SwissModel; we summarized the functional characterization of Lepidopteran SPRs and SPR-ligand interaction and then described the progress in the sex pheromone signaling pathways and metabotropic ion channel. Further studies are needed to work out the cryo-electron microscopy (EM) structure of SPR and the SPR-ligand docking pattern in a biophysical perspective, which will directly facilitate the understanding of sex pheromone signal transduction pathways and provide guidance in the sex lure technology in field pest control.

Climate change risk to pheromone application in pest management.

Since of the first sex pheromone and the adoption of pheromone in pest management, the global pheromone market size has grown to reach USD 2.4 billion per year in 2019. This has enabled the development of environmentally friendly approaches that significantly reduce the application of pesticides. Recently, there have been reports of the failure of various commercial codlemone: (E,E)-8,10-dodecadien-1-ol formulations used for monitoring the apple pest codling moth, Cydia pomonella (L.). This work was initiated to investigate factors behind the lack of efficacy of codlemone lure in the Northern Hemisphere (Germany) and Southern Hemisphere (New Zealand). We hypothesised that the observed failure could be due to two main factors: (a) a shift in the response of male codling moth to codlemone and (b) degradation of codlemone under field conditions that renders the lure less attractive. Field trial tests of various doses and blends containing minor pheromone compounds suggested no change in response of male codling moth. The addition of an antioxidant and a UV stabiliser to codlemone resulted in a significant increase in the number of males caught in Germany, but not in New Zealand. Mean maximum temperatures during the growing season since 2004 indicate a 3°C increase to 35°C in Germany, but just a 1.5°C rise to 30°C in New Zealand. Chemical analysis of the lures used in the field trials in Germany and New Zealand indicated more degradation products and reduced half-life of the lures in Germany compared with those in New Zealand. Heating codlemone lures to 32°C significantly reduced the number of males caught in traps and increased the isomeric and chemical impurities of codlemone compared with unheated lures. Our data provide the first evidence that climate change affects pheromone molecule stability, thus reducing its biological efficacy. Our finding suggests that climate change could be a general problem for chemical communication and, therefore, could affect the integrity of natural ecosystems.

Functional characterization of the epoxidase gene, Li_epo1 (CYP341B14), involved in generation of epoxyalkene pheromones in the mulberry tiger moth Lemyra imparilis

Epoxidation of alkenes derived from essential fatty acids is a key step in the biosynthesis of sex pheromones in moth species that utilize alkenyl sex pheromones. The position of the epoxy ring in the pheromone molecule differs depending on the species, thereby conferring diversities on sex pheromones. To date, only one pheromone gland (PG)-specific epoxidase, Hc_epo1 (CYP341B14), has been reported. Hc_epo1, which was identified from an arctiid moth Hyphantria cunea, catalyzes the epoxidation of a double bond at position 9 of the triene, Z3,Z6,Z9-21:H. In the present study, we investigated the PG-specific epoxidase from another arctiid, the mulberry tiger moth Lemyra imparilis, in order to verify whether cytochrome P450 in the CYP341B subfamily, to which Hc_epo1 belongs to, is responsible for the epoxidation of pheromone precursors at position 9 in moths other than H. cunea. A fragment of the Hc_epo1 homolog was amplified from cDNA prepared from the PG of L. imparilis by PCR with degenerate primers. The deduced amino acid sequence of the subsequently cloned homolog, Li_epo1, showed 88.5% identity to Hc_epo1. A functional assay using the Sf9 insect cell line–baculovirus expression system showed that Li_epo1 exhibited epoxidase activity with high selectivity to the double bond at position 9 of two trienes, Z3,Z6,Z9-21:H and Z3,Z6,Z9-23:H, precursors of epoxy diene sex pheromone components in L. imparilis.

The Molting Biomarker Metabolite N-acetylglucosamino-1,5-lactone in Female Urine of the Helmet Crab Telmessus cheiragonus.

N-acetylglucosamino-1,5-lactone (NAGL) is a molting biomarker in the blue crab Callinectes sapidus The concentration of this compound in urine is highest at the premolt stage. Since sexually mature premolt females release sex pheromone in their urine, NAGL is a candidate sex pheromone molecule in C. sapidus This compound has not been reported in other species. In the present study, we quantified the concentration of NAGL in the urine of the helmet crab Telmessus cheiragonus, using nuclear magnetic resonance spectroscopy, and found that the concentration increases toward the day of molting and decreases after molting. However, the total amount of NAGL collected from individual animals was greatest two to five days after molting, because the amount of urine collected was the lowest at the premolt stage, and it increased after molting. The highest median concentration of NAGL in T. cheiragonas was 29 μmol l(-1), which is 75% of the highest concentration reported in C. sapidus This is the first report of NAGL as a molting biomarker in a species other than C. sapidus We assume that NAGL is part of a pheromone bouquet in these two species.

Modeling and Regression Analysis of Semiochemical Dose–Response Curves of Insect Antennal Reception and Behavior

Dose-response curves of the effects of semiochemicals on neurophysiology and behavior are reported in many articles in insect chemical ecology. Most curves are shown in figures representing points connected by straight lines, in which the x-axis has order of magnitude increases in dosage vs. responses on the y-axis. The lack of regression curves indicates that the nature of the dose-response relationship is not well understood. Thus, a computer model was developed to simulate a flux of various numbers of pheromone molecules (10(3) to 5 × 10(6)) passing by 10(4) receptors distributed among 10(6) positions along an insect antenna. Each receptor was depolarized by at least one strike by a molecule, and subsequent strikes had no additional effect. The simulations showed that with an increase in pheromone release rate, the antennal response would increase in a convex fashion and not in a logarithmic relation as suggested previously. Non-linear regression showed that a family of kinetic formation functions fit the simulated data nearly perfectly (R(2) >0.999). This is reasonable because olfactory receptors have proteins that bind to the pheromone molecule and are expected to exhibit enzyme kinetics. Over 90 dose-response relationships reported in the literature of electroantennographic and behavioral bioassays in the laboratory and field were analyzed by the logarithmic and kinetic formation functions. This analysis showed that in 95% of the cases, the kinetic functions explained the relationships better than the logarithmic (mean of about 20% better). The kinetic curves become sigmoid when graphed on a log scale on the x-axis. Dose-catch relationships in the field are similar to dose-EAR (effective attraction radius, in which a spherical radius indicates the trapping effect of a lure) and the circular EARc in two dimensions used in mass trapping models. The use of kinetic formation functions for dose-response curves of attractants, and kinetic decay curves for inhibitors, will allow more accurate predictions of insect catch in monitoring and control programs.

(6R,10S)-Pallantione: The First Ketone Identified as Sex Pheromone in Stink Bugs

This work describes the structural elucidation of the sex pheromone of the soybean stink bug, Pallantia macunaima. The biological activity of the synthetic pheromone was demonstrated by behavioral and EAD experiments. Furthermore, the absolute configuration of the natural pheromone was determined as (6R,10S)-6,10,13-trimethyltetradecan-2-one. This is the first ketone identified as a male-produced sex pheromone in Pentatomidae, and the trivial name, pallantione, was assigned to this novel pheromone molecule.

Pheromonal bile acid 3-ketopetromyzonol sulfate primes the neuroendocrine system in sea lamprey.

BackgroundVertebrate pheromones are known to prime the endocrine system, especially the hypothalamic-pituitary-gonadal (HPG) axis. However, no known pheromone molecule has been shown to modulate directly the synthesis or release of gonadotropin releasing hormone (GnRH), the main regulator of the HPG axis. We selected sea lamprey (Petromyzon marinus) as a model system to determine whether a single pheromone component alters the output of GnRH.Sea lamprey male sex pheromones contain a main component, 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulfate (3 keto-petromyzonol sulfate or 3kPZS), which has been shown to modulate behaviors of mature females. Through a series of experiments, we tested the hypothesis that 3kPZS modulates both synthesis and release of GnRH, and subsequently, HPG output in immature sea lamprey.ResultsThe results showed that natural male pheromone mixtures induced differential steroid responses but facilitated sexual maturation in both sexes of immature animals (χ2 = 5.042, dF = 1, p < 0.05). Exposure to 3kPZS increased plasma 15α-hydroxyprogesterone (15α-P) concentrations (one-way ANOVA, p < 0.05) and brain gene expressions (genes examined: three lamprey (l) GnRH-I transcripts, lGnRH-III, Jun and Jun N-terminal kinase (JNK); one-way ANOVA, p < 0.05), but did not alter the number of GnRH neurons in the hypothalamus in immature animals. In addition, 3kPZS treatments increased lGnRH peptide concentrations in the forebrain and modulated their levels in plasma. Overall, 3kPZS modulation of HPG axis is more pronounced in immature males than in females.ConclusionsWe conclude that a single male pheromone component primes the HPG axis in immature sea lamprey in a sexually dimorphic manner.

Effect of two pyrazine-containing chemosignals on cells of bone marrow and testes in male house mice (Mus musculus L.)

Evolutionary conservative chemosignal 2,5-dimethylpyrazin that is pheromone in female mice has been shown to increase frequency of mitotic aberrations analyzed with aid of metaphasic and ana-telophasic analysis in bone marrow cells. Replacement of one of methyl radicals in the pheromone molecule by the carboxyl radical reveals specificity of action of the used derivative: the frequency of disturbances revealed only by the ana-telophasic analysis increases, whereas by the metaphasic analysis, no induction of disturbance is detected. In the sperm head abnormality test there is shown a rise of the anomalies by both compounds. Possible mechanisms of specific action of the tested substances on stability of genetic apparatus of the bone marrow dividing cells in the house mouse are discussed.

Stability of forest lepidopteran pheromones against environmental factors

A quantum-chemical study has been made of the interactions of pheromones of some lepidopteran forest pests (Siberian moth Dendrolimus superans sibiricus Tschetv and gypsy moth Limantria dispar L.) with components of the ambient air, as well as the effect of electromagnetic radiation on the pheromones. It is found that the reactions of pheromones with substances contained in the forest air are irreversible and proceed with liberation of heat. Electromagnetic radiation quite strongly affects the structure of pheromones, whereby the pheromone molecule is activated and can readily enter into reactions.

Comparisons of simple and complex coacervations for preparation of sprayable insect sex pheromone microcapsules and release control of the encapsulated pheromone molecule

With dodecanol (C12OH) as a model molecule of insect sex pheromone as core material, natural polymers gelatin (GE) and acacia gum (AG) as wall materials, microcapsules aiming to be a sprayable environment-friendly pesticide were prepared via GE simple coacervation and complex coacervation of GE and AG. C12OH encapsulation in complex coacervation was higher than those in simple coacervation. Its encapsulation was enhanced with increase in wall material cross-linking. C12OH release revealed that samples from simple coacervation reached their end in 7 days, whereas those from complex coacervation manifested a quick release followed by a constant release. With increase in wall material cross-linking, the release was slowed down. SEM observation confirmed that core-shell morphology existed in the capsules.

Modulation of Gonadotrophin‐Releasing Hormone Pulse Generator Activity by the Pheromone in Small Ruminants

In small ruminants, such as goats and sheep, a primer pheromone produced by males induces an out-of-seasonal ovulation in anoestrous females, a phenomenon known as the male effect. The male effect is unique in that an external chemical stimulus can immediately modulate the activity of the hypothalamic gonadotrophin-releasing hormone (GnRH) pulse generator. We have established a monitoring method of the GnRH pulse generator activity in Shiba goat. Using this method as a sensitive bioassay to assess the male effect pheromone activity, we have shown that the male effect pheromone is synthesised in an androgen-dependent manner in the sebaceous glands or their vicinity in specific body regions in goats. Although chemical identity of the pheromone is yet to be determined, analyses of male goat hair extracts by gas chromatography fractionation suggest that the male effect pheromone is a volatile substance with relatively small molecular weight. From morphological and molecular biological studies in goats, it is suggested that the pheromone molecule is detected by a member of the V1R family located on both the olfactory neurones and the vomeronasal sensory neurones, and the pheromone signal is conveyed to the medial nucleus of amygdala via the main olfactory and vomeronasal pathways and, subsequently, to the hypothalamic GnRH pulse generator to enhance its activity.

PREPARATION OF MICROCAPSULES WITH ENCAPSULATED DODECANOL BY COMPLEX COACERVATION OF POLY［2-(METHACRYLOYLOXY)-ETHYLTRIMETHYL AMMONIUM CHLORIDE］ AND ACACIA

将聚［2-(甲基丙烯酰氧)乙基三甲基氯化铵］(PMTC)和阿拉伯胶(GA)在一定条件下进行了复凝聚，并对影响复凝聚实验的壁材配比、壁材浓度、离子强度等因素进行了考察.实验结果表明，PMTC与GA配比为1/3.22，壁材总浓度为4％时复凝聚效率最高；体系中不同浓度的氯化钠的存在会对复凝聚起到不同程度的抑制作用.在实验确定的最佳复凝聚条件下以有机小分子化合物十二醇作为芯材进行了包覆，制备了不同壁芯比例的微胶囊.对微胶囊的包覆率及载药量进行了测量，并对它们的释放行为进行了考察.包覆有十二醇的复合微胶囊大小一般在几微米.随着壁材与芯材比例的增大，胶囊载药量逐渐降低，微胶囊释放十二醇的速率明显变小，但包覆率却无明显变化规律.

Chemistry of microbial signal compounds

The ComX pheromone is a post-translationally modified oligopeptide that stimulates natural genetic competence controlled by quorum sensing in Bacillus subtilis. The structure of the ComXRO-E-2 pheromone produced by strain RO-E-2 was determined. In the pheromone molecule, a geranyl group is bound to the tryptophan residue, which results in the formation of a tricyclic ring structure. Molds in the genus Phytophthora are some of the most destructive phytopathogens known. An important biological event in Phytophthora is sexual reproduction. A factor known as hormone α1 is secreted by the A1 mating type, and this induces the formation of oospores in the A2 mating type. The structure of hormone α1 is a C-20 linear isoprenoid and the absolute stereochemistry was determined by chemical synthesis.

Neurogenic and Neuroendocrine Effects of Goldfish Pheromones

Goldfish (Carassius auratus) use reproductive hormones as endocrine signals to synchronize sexual behavior with gamete maturation and as exogenous signals (pheromones) to mediate spawning interactions between conspecifics. We examined the differential effects of two hormonal pheromones, prostaglandin F(2alpha) (PGF(2alpha)) and 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) on neurogenesis, neurotransmission, and neuronal activities, and on plasma androstenedione (AD) levels. Exposure to waterborne PGF(2alpha) induced a multitude of changes in male goldfish brain. Histological examination indicated an increase in the number of dividing cells in male diencephalon (p < 0.05, Kruskal-Wallis test). Real-time quantitative PCR tests showed elevated levels of transcripts for the salmon gonadotropin-releasing hormone (GnRH) in the male telencephalon and cerebellum (p < 0.005, one-way ANOVA) and for ChAT (choline acetyltransferase) in the male vagal lobe and the brainstem underneath the vagal lobe (p < 0.05, one-way ANOVA). Therefore, PGF(2alpha) seemed to modulate male brain plasticity that coincided with behavioral changes during spawning season. Exposure to waterborne 17,20beta-P, however, increased circulatory levels of immunoreactive AD in males and the transcripts of androgen receptor and cGnRH-II (chicken-II GnRH) in the female cerebellum (p < 0.05, one-way ANOVA). PGF(2alpha) and 17,20beta-P thereby seemed to act through distinct pathways to elicit different responses in the neuroendocrine system. This is the first finding that links a specific pheromone molecule (PGF(2alpha)) to neurogenesis in a vertebrate animal.

Theoretical analysis of the electronic properties of the sex pheromone and its analogue derivatives in the female processionary moth Thaumetopoea pytiocampa

In the present work, the distribution of the electronic charge density of the natural sex pheromone, the ( Z)-13-hexadecen-11-ynyl acetate, in the female processionary moth, Thaumetopoea pytiocampa , and its nine analogue derivatives was studied within the framework of the Density Functional Theory and the Atoms in Molecules (AIM) Theory at B3LYP/6-31G∗//B3LYP/6-31++G∗∗ level. Additionally, molecular electrostatic potential (MEP) maps of the previously mentioned compounds were computed and compared. Furthermore, the substitution of hydrogen atoms from the methyl group in the acetate group by electron withdrawing substituents (i.e., halogen atoms) as well as the replacement effect of hydrogen by electron donor substituents (+I effect) as methyl group, were explored. The key feature of the topological distribution of the charge density in analogue compounds, such as the variations of the topological properties encountered in the region formed by neighbouring atoms from the substitution site were presented and discussed. Using topological parameters, such as electronic charge density, Laplacian, kinetic energy density, and potential energy density evaluated at bond critical points (BCP), we provide here a detailed analysis of the nature of the chemical bonding of these molecules. In addition, the atomic properties (population, charge, energy, volume, and dipole moment) were determined on selected atoms. These properties were analyzed at the substitution site (with respect to the natural sex pheromone) and related to the biological activity and to the possible binding site with the pheromone binding protein, (PBP). Moreover, the Laplacian function of the electronic density was used to locate electrophilic regions susceptible to be attacked (by deficient electron atoms or donor hydrogen). Our results indicate that the change in the atomic properties, such as electronic population and atomic volume, are sensitive indicators of the loss of the biological activity in the analogues studied here. The crucial interaction between the acetate group of the natural sex pheromone and the PBP is most likely to be a hydrogen bonding and the substitution of hydrogen atoms by electronegative atoms in the pheromone molecule reduces the hydrogen acceptor capacity. This situation is mirrored by the diminish of the electronic population on carbon and oxygen atoms at the carbonylic group in the halo-acetate group. Additionally, the modified acetate group (with electronegative atoms) shows new charge concentration critical points or regions of concentration of charge density in which an electrophilic attack can also occur. Finally, the use of the topological analysis based in the charge density distribution and its Laplacian function, in conjunction with MEP maps provides valuable information about the steric volume and electronic requirement of the sex pheromone for binding to the PBP.

Olfactory Discrimination among Sex Pheromone Stereoisomers: Chirality Recognition by Pink Hibiscus Mealybug Males

Our previous field studies suggested that the two chiral centers in the sex pheromone of pink hibiscus mealybug, Maconellicoccus hirsutus, could elicit different male responses. The chiral center in the acid moiety of the pheromone seemed to be more critical than the alcohol portion of the pheromone molecule for attractiveness. The objective of the current study was to test this hypothesis by deploying stereoisomeric blends in pheromone traps. Captures of male M. hirsutus showed that pheromone with the naturally occurring (R)-maconelliyl (S)-2-methylbutanoate and (R)-lavandulyl (S)-2-methylbutanoate [R-S configuration] was most attractive and that pheromone with the unnatural S-S configuration was less attractive. In addition, the RS-R blend (containing R-R and S-R stereoisomers) yielded captures of male M. hirsutus that were comparable to blank controls, and an inhibitory effect was observed when R-R and S-R were combined with naturally occurring R-S blend. These results suggest a unique chirality recognition mechanism; olfactory discrimination among different pheromone stereoisomers depends upon both asymmetric centers. The S configuration on the acid moiety elicits attraction, whereas the R configuration induces inhibition. However, the attractive activity shows some degree of tolerance toward chirality change in the alcohol portion of the pheromone molecules.

Structural Consequences of the pH-induced Conformational Switch in A. polyphemus Pheromone-binding Protein: Mechanisms of Ligand Release

Olfaction in moths is one of the most impressive examples of chemical communication found in nature for its exquisite sensitivity and selectivity. Pheromone-binding proteins (PBPs), present in the antennae of male moth and other insect species, bind the hydrophobic pheromone molecules and transport them to the G protein-coupled olfactory receptor proteins. The targeted delivery of these non-polar ligands to membrane-bound receptors involves ligand release on or near the target cell membranes, the molecular details of which are still not well understood. The PBP from the giant silk moth Antheraea polyphemus (ApolPBP) binds acetate pheromone only at pH above 6.0, and its structure at pH 6.3 has been determined previously. Here we report the solution NMR structure of ApolPBP at the acidic pH 5.2. Comparison of the present structure to that at neutral pH reveals the details of the pH-induced conformational changes and provides mechanistic clues for ligand release at acidic pH. The ApolPBP pH-induced structural change is quite different from that observed for alcohol binding Bombyx mori PBP (BmorPBP), where the C-terminal segment folds into a helix and occupies the ligand binding cavity. We observe a reorientation of helices alpha1, alpha3, and alpha4 at acidic pH caused by protonation of His69, His70 and His95 in the interior. This provides the driving force behind the opening of the ligand binding cavity and the release of the pheromone molecule from its carrier protein near the membrane.

Regionally Specific Occurrence of an Active Sodefrin Variant in the Red‐Bellied Newt

Sodefrin (SIPSKDALLK) is a female-attracting pheromone that is secreted by the abdominal gland of the male red-bellied newt. We found that mRNA encoding a sodefrin variant, [Val(8)] sodefrin, is expressed exclusively in specimens captured in the Nara area of Japan. The synthetic peptide was tested for its activity. It attracted females from Nara, but not those from other regions, suggesting that there is a geographic variation in the pheromone molecule and in the responsiveness to the pheromone. Employing an abdominal gland extract and synthetic substrates, the possibility of generation of the putative pheromone, [Val(8)] sodefrin, from the precursor molecule was demonstrated.

Characterization of the Primer Pheromone Molecules Responsible for the 'Male Effect' in Ruminant Species

Pheromones are chemical signals used among consepcifics for social interactions such as sex attraction, mate selection, modulation of neuroendocrine function, and individual identification (Vandenbergh, 1994; Singh, 2001; Halpern and Martinez-Macros, 2003). The male effect is a representative example of such pheromonal actions in goats and sheep, in which exposure to the male pheromone accelerates the pulsatile gonadotropin-releasing hormone (GnRH)/luteinizing hormone release and thereby the ovarian activity in the female (Schinkel, 1954; Martin et al., 1986). Several attempts have been made to isolate pheromone molecule(s) responsible for the male effect using the induction of ovulation (Knight and Lynch, 1980) or the stimulation of LH release (Claus et al., 1990; Cohen-Tannoudji et al., 1994) as indices for the pheromone activity. These studies found that the male hair but not urine contained the pheromone activity. Therefore, chemical constituents in the male hairs were extracted and processed for further fractionation and analysis. Although the pheromone activity was observed in the fractions of initial steps, it eventually disappeared as the fractionation proceeded. Thus, the chemical identity of the primer pheromone has not yet been determined so far. A major problem of those studies appears to reside in the bioassay systems they employed, which required several time consuming steps to determine whether the sample in question contained the pheromone or not. To overcome this matter, we have established a bioassay system to assess the pheromone activity in a real-time manner with high sensitivity. Utilizing this system, we have been conducting a series of experiments aiming at elucidation of production mechanism and the isolation of chemical molecule (s) of pheromone responsible for the male effect in the Shiba goat, a Japanese indigenous breed.