Buck Odor Production in the Cornual Gland of the Male Goat, Capra Hircus– Validation with Histoarchitecture, Volatile and Proteomic Analysis

Indian Journal of Biochemistry & Biophysics Vol. 55, June 2018, pp. 183-190

Buck odor production in the cornual gland of the male goat, Capra hircus– Validation with histoarchitecture, volatile and proteomic analysis

Devaraj Sankarganesh1,#, Rajamanickam Ramachandran1,2, Radhakrishnan Ashok1, Veluchamy Ramesh Saravanakumar3, Raman Sukirtha1, Govindaraju Archunan4* & Shanmugam Achiraman1,4* 1Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India 2Department of Microbial Biotechnology, Bharathiar University, Coimbatore-641 046, Tamil Nadu, India 3Department of Livestock Production and Management, Veterinary College and Research Institute, Namakkal-637 002, Tamil Nadu, India 4Center for Pheromone Technology, Department of Animal Science, Bharathidasan University, Tiruchirappalli- 620 024, Tamil Nadu, India

Received 28 January 2018; revised 14 May 2018

In many animals, glandular secretions or pheromones that possess biological moieties contain messages encoded by the intrinsic smell. In male goats, the cornual gland (a sebaceous gland), may synthesize and excrete relevant chemical components that are responsible for the ‘buck effect’. To test this, cornual glands from freshly-slaughtered male goats (N=6) were subjected to histoarchitecture analysis, to infer about the structural alignment, to the GC–MS analysis for volatile compounds and to SDS–PAGE for protein profiling followed by MALDI-TOF to characterize specific protein bands. The gland possesses sebum, vacuoles and hair follicles inferring its capability to synthesize and extrude the scent. We found 14 volatiles in GC–MS analysis, in which 1-octadecanol might be a putative pheromone of buck odor. We identified seven different proteins in SDS-PAGE. Two proteins, 28 and 33 kDa, were highly matched with DNA mismatch repair protein and Abietadiene synthase, respectively, as inferred from MALDI-TOF. Conclusively, the volatiles identified in the cornual gland suggest that the structural microelements of the gland may synthesize (sebum and vacuoles) and release the key volatiles through the hair follicles. The volatile(s) thus produced in male goats either solely or synergistically may confer the buck odor.

Keywords: GC–MS, Goaty odor, MALDI-TOF, Putative pheromone, Scent gland, Sebum

Physiological status of animal can be assessed by synchronization of estrus by increasing the LH pulse conspecifics through a variety of scent sources, rate and leads to ovulation (buck effect). Perhaps all including glandular secretions. Mammalian the stimuli (odor, sight, sound, and touch) are pheromones are characterized by multiple compounds mutually involved; odor seems to be a key element in used in intra-specific communication1. In goats, ‘buck regulating the buck effect2. Hamada et al.3 utilized the odor’ is believed to be produced in the skin/hair/gland hair samples of males as an odor source and exposed of intact males and has the capacity to synchronize/ it to the female goats for a short period and found an regulate the reproductive cycle and ovulation in the increase in GnRH pulses in females. Subsequently, female. The introduction of sexually naïve bucks Ichimura et al.4 exposed the female goats with the to a group of non-ovulating females results in pooled hair samples of males in a muzzle-fitted mask relatively for a longer period (4 h) and observed —————— *Correspondence: greater GnRH pulses. The compound responsible for 5 Phone (M): +91-9894835753 (S. A.); +91-9443922228 (G. A.) the buck odor was identified as 4-ethyloctanoic acid . E-mail: [email protected] (S. A.); [email protected] (G. A.) Lancker et al.6 reported a sebaceous gland #Present address: Department of Biotechnology, School of Bio underneath the horns of the male goats. Earlier, the and Chemical Engineering, Kalasalingam Academy of Research gland was swabbed and volatile compounds were and Education, Krishnankoil-626 126, Tamil Nadu, India identified in an attempt to validate the buck effect, but Abbreviations: GC–MS, Gas Chromatography-Mass Spectrometry; no conclusive information was derived as decades-old SDS-PAGE, Sodium dodecyl sulfate-Polyacrylamide gel chemical separation (chromatography) techniques electrophoresis; MALDI-TOF/MS, Matrix-assisted laser 7 desorption/ionization-time of flight/Mass Spectrometry; PBS, were adopted . Interestingly, the upper dermal layer Phosphate-buffered saline; MMR protein, Mismatch repair protein extracts of sebaceous glands from intact and castrated 184 INDIAN J. BIOCHEM. BIOPHYS., VOL. 55, JUNE 2018

plus testosterone administered goats were shown to clearly indicate that hair/skin samples of head area induce greater multiple unit activities in female contain compounds of goaty odor, which is goats8. Further, it is also demonstrated that the extract presumably due to the presence of sebaceous glands of cutaneous gland possesses active ingredients to (cornual gland). However, the study of the cornual 9-11 11 produce buck odor . Remarkably, Murata et al. gland in the perspectives of histology, and volatile identified a single olfactory molecule (4-ethyloctanal) analysis in the extracts of the gland is not performed, that is responsible for the elicitation of buck effect by which may further shed light on buck effect. confirming the multiple unit activity and induction of Therefore, we embarked a study on the cornual gland LH pulses in female goats. However, hitherto no to 1) Confirm the secretory and excretory nature of study was performed in the extracts of the cornual the gland by subjecting it to histological analysis, 2) gland to identify the complete volatiles. Detect the complete volatiles (putative pheromones) In many animals, the scent gland and the volatile in the extract of the gland by GC–MS analysis, and 3) compounds produced in it have pivotal roles in To elucidate the protein profile to look for the communication among conspecifics. For instance, presence of carrier proteins, if any. Atta laevigata, an ant, has a mandibular gland (modified scent gland) and the concentration of the Materials and Methods compound it synthesizes varies in relation to the Test animals and collection of gland samples castes12. In male lizards, the femoral gland with Adult male goats (N=6), Capra hircus fed with granular cells is the major source of pheromones13. standard ration and water ad libitum were used for the The mammary gland of the rabbit secretes milk that present study. The inclusion criterion was sexually contains 2-methyl but-2-enal, which is believed to be intact male goats and they were not castrated. The a mediator of mother-young interaction14. The goats were sacrificed at the slaughter house during preputial gland of rodents has been reported to play a April-May when their age was <24 months, and key role in communication between sexes. For 12 gland samples (2 from each) were obtained. The instance, in an earlier investigation, we reported that gland was located as described previously6. The hairs the clitoral gland of female laboratory rats contains an in the caudomedial border beneath the horns were estrus-specific compound which could be a mediator shaved and the gland was located as a distinct 15 of sexual communication . In addition, synthesis of oval-shaped structure with 1–3 cm in length and the key constituents of the preputial gland of male rat 0.5–1.5 cm wide. We used one gland from each goat is testosterone-dependent, which indicates that there is for extract preparation by homogenizing with PBS. a relationship between glandular secretions and sexual This extract was used for volatile analysis in GC–MS 16 status of animals . and remnant of the extract was processed for In hoofed mammals, (e.g., deer) the preorbital SDS–PAGE analysis. Another gland from each goat gland is a good source of chemical compounds that was subjected to histological sectioning and facilitate chemo-communication, and some of these representative of single gland structure was shown in compounds have been implicated in the expression of results. The study does not involve live animals, and dominant status by a particular male animal the glands were collected from the monitored animals 17 in the colony . Considerable difference in the after sacrification at the slaughter house. histoarchitecture of preorbital gland has been shown between territorial and non-territorial Indian Histoarchitectural analysis blackbuck18,19. In elephants, the temporal gland The glands were dissected and fixed immediately secretes a fairly good quantity of musth, which is in 10% neutral buffered formalin, and subsequently reported to be androgen-dependent20. The axillary embedded in paraffin. The sections (4 µm) were gland secretion is one of the important sources of made, and stained with haematoxylin and eosin. The human pheromones. The secretory material has a stained sections on glass slides were examined under peculiar smell due to microbial action and confers the a light microscope (Wetzlar Hund, Germany) and message of maleness21. photographs were captured with real-time Considering the large population size and great interpretations by a veterinary anatomist. diversity in mammals, reports are fragmentary in Sample preparation support of the glandular sources performing roles in The glands from six goats were sliced into small chemical communication. In goat, previous reports pieces using sterilized blades, which were SANKARGANESH et al.: BUCK ODOR PRODUCTION IN THE CORNUAL GLAND OF THE MALE GOAT 185

homogenized using a sterilized homogenizer De-staining and in-gel digestion with PBS (pH 7.2). A part of the homogenate The protein bands of interest were excised and the (approximately 5 mL) was extracted with gel plugs were de-stained separately using 100 µL of dichloromethane in 1:1 ratio in a sterilized glass vial. 25 mM NH4HCO3 in 50% (v/v) acetonitrile (1:1) by Briefly, the extract was vortexed and kept in the incubation at 37°C for 30 min. Each gel plug was refrigerator to allow distinct separation of layers of sliced into small cubes, and placed in 1.5 mL the extract. After 30 min, the solvent layer (crystal- Eppendorf tube. After drying in a Speed–Vac like) was transferred to GC–MS vial using a Pasteur (Savant), the gel was incubated in 100 µL of 2% pipette and used for analysis of volatile compounds in -mercaptoethanol/25 mM NH4HCO3 for 20 min at GC–MS. room temperature. The same volume of 10% 4-vinylpyridine in 25 mM NH4HCO3/50% acetonitrile Gas chromatography–mass spectrometry was added for cysteine alkylation. After 20 min, the The final extract was filtered with Whatman filter gel was soaked in 1 mL of 25 mM NH4HCO3 for paper (Grade 1, 11 µm pore size, Merck Biosciences) 10 min, dried and incubated overnight with 25 mM before injected into GC–MS (QP-2010 plus, NH4HCO3 containing 100 ng of modified trypsin Shimadzu, Japan). Two micro-liters of the pooled (Promega). The tryptic digest was removed from the extract was injected into the GC–MS using the gel and the proteins were extracted first with 300 µL auto-injector. The injector temperature was 200°C and of 25 mM NH4HCO3 and then in 50% acetonitrile. operated in split mode. The GC–MS was fitted with a The two fractions were pooled and dried in a Speed– 30 m capillary column with a film thickness of Vac and then stored at −20°C for further analysis. 0.25 µm (30 m × 0.2 mm i.d., coated with UCON HB When resumed, each fraction was re-suspended in 2000). The temperature regimens were as follows; 0.1% formic acid immediately before use24. initial oven temperature, 40°C for 10 min; ramp 1 to 150°C at a rate of 5°C/min; ramp 2 to 230°C at a rate MALDI–TOF MS analysis of 10°C/min and finally held at 230°C for 4 min. The The MALDI-TOF MS data were acquired using an detection accuracy was 1 ng/peak. The mass Ultraflex TOF/TOF spectrometer (Bruker Daltonics, spectrometer was operated in electron ionization Billerica, MA, USA), equipped with 50 Hz pulsed mode at 70 eV, and ammonia was used as carrier gas nitrogen laser (337 nm), operated in reflectron mode at a flow rate of 1.0 mL/min. The identification of (positive ion) at 25 kV accelerating voltage with unknown compounds was made by probability-based 90-ns time delay. External calibration was done using matching, using WILEY RegistryTM, NIST05, and peptide I calibration standards (angiotensin II, NIST05s based library. angiotensin I, substance P, bombesin, ACTH clip 1–17 and 18–39, and somatostatin 28) with molecular SDS–PAGE analysis masses ranging 1000–3200 Da. The samples were The remnant of the gland extract (homogenized prepared by mixing an equal amount of peptide using PBS) was centrifuged at 10000 rpm for (0.5 μL) with dihydroxybenzoic acid/α-cyano-4- 10 min at 4°C (Remi Cooling Centrifuge). The hydroxycinnamic acid (CHCA) saturated with 0.1% resulted pellet was discarded and the supernatant was trifluoroacetic acid (TFA) and acetonitrile (1:1) as transferred to another tube. Primarily, the protein matrices. The mass spectrum of the 33 and 28 kDa quantity in the supernatant was estimated adopting proteins was obtained and the mono-isotopic masses Bradford method22 by reading the formed blue color of the protein spectrum were analyzed. The masses solution in a spectrophotometer at 595 nm. Sixty below 50 m/z were not considered due to interference micrograms of protein sample were taken and from the matrix. processed with sample loading dye and used in SDS–PAGE analysis. The gel was stained with Database analysis Coomassie brilliant blue and de-stained (ethanol, The spectra were processed using Flex analysis acetic acid, and water) to visualize the bands23. We software. Database search was accomplished with the have used molecular weight marker of medium range assigned masses of the monoisotopic peptide. Mascot (Genei, Bangalore, India, Cat. No: 105979), which search engine (Matrix Science, London, UK) helped us to speculate the molecular weight of the (http//www.matrixscience.com) was utilized for the proteins present in our sample. protein identification. Mascot analysis was carried out 186 INDIAN J. BIOCHEM. BIOPHYS., VOL. 55, JUNE 2018

using carbamidomethyl as fixed modification and Volatile compounds in the cornual gland extract and their oxidation (M) as variable modification. The search features as revealed in the GC–MS analysis was made under the taxonomy of the Eukaryota for Fourteen volatiles were identified in the cornual 33 kDa protein and under all taxonomical entries for gland extract, among which most of the compounds 28 kDa protein. The score value was checked for the were alkanes and alkenes whereas, some were level of significance and matched sequences (bold and alcohols. The molecular weights of the compounds underlined) were identified. were between 84 and 280 Da. There were 4–20 carbon atoms, and 3–40 hydrogen atoms in the Results compounds (Table 1; Fig. 2). Structural organization of cornual gland as revealed by histoarchitecture analysis Proteins in the cornual gland extract as revealed in the The gland is located close to the apical region of SDS–PAGE analysis the dermal papilla. It opens at the dermal pore and is The protein extract of the gland was separated in surrounded by the roots of the hair follicles. The 12% SDS–PAGE, which revealed the presence of surrounding area is filled with smooth muscle fibers, seven different protein fractions, and their molecular running at a different direction, i.e., cross, oblique and weights ranged from 15 to 58 kDa. Among the longitudinal. The contraction of the smooth muscle different proteins, the intensity of 48 kDa protein fiber between the lobules and the fibers located fraction was the highest (Fig. 3). around the gland could be involved in the extrusion of Table 1 — List of compounds identified in the cornual gland sebum. Acini are serous, i.e., formed of small lobules, extract of male goat, Capra hircus and the acinar gland is lobulated (Fig. 1). Compound name Molecular Molecular formula weight (Da)

Cis-1-Chloro Buten-3-yne C4H3Cl 86

1-Cyclopropyl octane C11H22 154

Tri cyclohexane C6H12 84

Alpha Dodecene C12H24 168

5-Octadecene C18H36 252

1,2 Hydroxyethoxy tridecane C13H28 184

2,4-bis 1,1 dimethyl ethyl phenol C6H6O 94

1-Pentadecene C15H30 210

5-Eicosene C20H40 280

2,3 Dimethyl hexane C8H18 114

1,2 Benzenedicarboxylic acid C9H8O 164

2-Methyl 2-Butenal C5H8O 84

1-Hepten-5-one C7H12O 112

1-Octadecanol C18H38O 270

Fig. 1 — Histoarchitecture of the cornual gland of male goat (10 & 40 X). The picture depict the presence of Vacuoles (A), Fig. 2 — GC–MS chromatogram of male goat cornual gland Hair follicles (B), and Duct (C) in the gland extract SANKARGANESH et al.: BUCK ODOR PRODUCTION IN THE CORNUAL GLAND OF THE MALE GOAT 187

Fig. 3 — SDS–PAGE profile of male goat cornual gland extract [L1: Marker and L2: Cornual gland extract]

Fig. 4 — Mass Spectra of 28 kDa protein obtained from MALDI– Identification of the protein as revealed in MALDI–TOF TOF. Among the different protein fractions, two protein A. Number in the mass spectrum gives precise m/z (M+H) values fractions (at 28 and 33 kDa) were subjected to for the detected peptide ion signals. MALDI–TOF analysis, which revealed high score B. The matched 22% sequence coverage shown in bold and underlined. matching with DNA mismatch repair protein and abietadiene synthase, respectively, in MASCOT analysis. The sequence coverage for DNA mismatch repair protein was 22% and that for abietadiene synthase was 20% (Figs. 4 & 5, matched sequences are indicated bold).

Discussion The histological technique was adopted to have an idea about the structural organization of the cornual gland and the observation confirmed that the cornual gland is a sebaceous gland that produces sebum. Previous studies have shown that rat preputial gland produces volatile compounds and also some proteins involved in communication10. It is an established fact that sebaceous gland produces sebum, which is an oily and waxy substance as documented in the humans25. According to Abbasi et al.26 the interdigital gland of sheep produces sebum and is discharged by the hair follicles present adjacent to the lobules. We also made a similar finding, and the cornual gland is Fig. 5 — Mass Spectra of 33 kDa protein obtained from MALDI- formed of numerous lobules, located adjacent to the TOF. A. Number in the mass spectrum gives precise m/z (M+H) values hair follicles indicating that sebum is produced and it for the detected peptide ion signals. is extruded via the hair follicles. In the deer, the fluid B. The matched 20% sequence coverage shown in bold and secretion release is visible, whereas in the goat, the underlined. 188 INDIAN J. BIOCHEM. BIOPHYS., VOL. 55, JUNE 2018

extrusion of the secretory material is not visible, but body secretions (urine and feces) of female goats28, one can sense the pungent odor of the glandular and urine of male goats (unpublished data). Therefore, secretion, which indicates that the secretion is we strongly conclude that 1-octadecanol is a gland- released as a volatile substance. However, on several specific volatile produced only in the cornual gland of occasions, the gland is ruptured by hitting against the intact male goats. wall of fence especially, when the buck was Most of the compounds identified in the present encountered with a female goat in estrus study are not new but have already been reported to (Sankarganesh, personal communication). Thus, it is act as pheromones in other animals and insects. The comprehensive that the gland does secrete a material, compound 2-methyl-2-butenal, identified in the and when the buck desires to mate with a female in cornual gland has been earlier reported in rabbit’s estrus, the gland is ruptured and the secretion milk, where it facilitates typical nipple searching discharged as a volatile substance. behaviour in rabbit pups29. Characterization of the A pioneering study on goat scent gland suggested other compounds also revealed most of them to be that cornual gland lacks the capacity to produce any pheromones but the exact role has not been evaluated. pheromones6; however, the study did not speak about The database available on the website (pherobase) volatiles. To validate and support our hypothesis, we gives adequate details about each of the pheromonal carried out some preliminary studies so as to conclude compounds. Thus, some of the compounds identified that cornual gland is one of the major scent glands in the present study (e.g., 5-Eicosene) have been and may be involved in buck odor production. Earlier, reported in the pherobase. it was reported that the scent gland of insects has The compounds produced from scent sources are distinctive roles in producing the compounds, mostly generally volatile in nature and, therefore, they are volatiles, and these compounds have been connected associated with carrier molecules which helps in with eliciting behavioural responses. In this context, sustained release of the signal. Flower30, in his we analyzed the volatile portion of the gland and review, has elucidated the structural and functional found 14 volatile compounds through GC–MS aspects of the lipocalin superfamily proteins, to which analysis, of which most of the compounds were the carrier proteins generally belong. A number of alkanes, but few belonged to alkenes, aldehyde, original studies have explained the possible role of ketone, and alcohol groups. The compounds under the lipocalin proteins which are related to chemical class alkanes, isoalkanes, alcohols, ketones, communication. However, in goats, the presence of carboxylic acids, saturated- and unsaturated- carrier protein in any body secretions or scent sources aldehydes have been reported in interdigital secretions was not evaluated. Therefore, we looked for carrier and suggested to have a role in the expression of the molecules that would bind and deliver volatile dominant status27. Therefore, we have reason to compounds. Seven protein bands, in the molecular believe that the volatile compounds identified in the cornual gland of goat in the present study appear to weight ranging from 15 to 58 were identified. To have some crucial role in goat communication. Thus, elucidate the functional aspect of the proteins, we we propose that the compounds identified in the subjected two of the proteins to MALDI–TOF present study are putative pheromones that might be analysis that were identified as DNA mismatch repair released via hair follicles. Interestingly, Murata et al.11 protein and abiedatiene synthase. However, these two have shown that the head skin of male goats contains proteins are not carrier proteins, but appear to be a compound, 4-ethyloctanal, which they believed to regular proteins and have other roles. be involved in buck effect. However, in our study, we DNA mismatch repair protein (MMR protein) has found 1-octadecanol in the glandular extract. It could been reported in the sebaceous gland of other animals, be postulated that there may be a possible where its function is crucial in inhibition of tumor derivatization of 4-ethyloctanal to 1-octadecanol formation since the absence of this protein have been while released through the hair follicles. In addition, linked to Lynch syndrome31. Tome et al.32 identified the other compounds belonged to ketone, alkanes, and DNA mismatch repair protein in various tissues of alkenes reported by Murata et al.11 are similar to our mice and found its concentration to be higher in the findings. It is important to note from the present study proliferative tissues than non-proliferative tissues. As that 1-octadecanol is present only in the glandular cornual gland is one among the proliferative tissues, extract of intact male goat and not found in other the presence of MMR protein must associate with SANKARGANESH et al.: BUCK ODOR PRODUCTION IN THE CORNUAL GLAND OF THE MALE GOAT 189

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