Cloning and sequencing of α-2u globulin of rat preputial gland to assess its longevity in the context of developing an effective rodent trap

α-2u globulin, a pheromone binding protein found majorly in the preputial gland, was reported to involve in chemo-communication of rats. The biosynthesis of this pheromone binding protein is under complex multihormonal control and its regulation takes place at transcription level. Assessing and increasing the longevity of this protein may retain the volatility of pheromone. So far nearly 20 isoforms of α-2u globulin in rat have been studied. The present study was aimed to extend the longevity of pheromone compound by cloning and sequencing of the mRNA which codes for α-2u globulin in the preputial gland of Rattus novergicus. Unexpectedly, this study resulted in a new isoform, which is similar in function with α-2u globulin protein with some different exons removed. Further analysis with this isoform may pave a way for rodent pest management.

Identification of pheromone-carrying protein in the preorbital gland post in the endangered Indian male Blackbuck Antelope cervicapra L.

In mammals, a low molecular mass protein (17-20 KDa) reported from the pheromone sources such as urine, saliva, glandular secretion, etc., as ligand-carrier (pheromone carrier) has been associated with chemo-communication. Since the preorbital gland post is one of the major pheromone sources in Indian Blackbuck, an endangered species, we assumed thta it possibly contains low molecular mass protein for chemical communication. Hence, we investigated the preorbital gland post in territorial and non-territorial male blackbucks for such low molecular mass proteins adopting SDS-PAGE and LC-MS/MS analysis. The total content of protein was higher in the post of territorial males than non-territorial males of adult and sub-adult. In fact, the protein profiles such as 17, 21, 25, 42 and 61 kDa were noted in the gland secretion of territorial and non-territorial males. The intensity of the 17 kDa protein band was higher in territorial males than non-territorial males. In-gel trypsin digestion of the 17 kDa band was processed and subjected to LC-MS/MS and SEQUEST analyses. The results of LC-MS/MS and SEQUEST search showed the presence of α2u-globulin in the 17 kDa band. In addition, the identified α2u-globulin sequence possessed GDW residues, which are the characteristic signature for lipocalin family. Since the α2u-globulin has been reported from the pheromone-carrying proteins in some mammals, this protein may carry the volatiles (pheromone compounds) in male Blackbucks preorbital gland to evoke the scent marking for maintaining territoriality (home range) and attraction towards female, through the secretion of glandular protein.

Stud male-originating chemosignals: A luteotrophic agent.

The chemosignals from mating male are found to be responsible for protecting his coital partner against pregnancy failure induced by strange male or food-deprivation. The stud male pheromone not only provides luteotrophic support in female of vulnerable condition but also exerts luteotrophic effect in pregnancy-blocked females by inducing pseudopregnancy. The luteotrophic stimulus rendered by stud male to prevent pregnancy failure is mediated through the main olfactory system, and not through the accessory olfactory system, since the accessory olfactory system is primarily involved in perceiving the luteolytic stimulus produced from strange male for causing pregnancy failure. It has been shown that pericopulatory period seems to be crucial in females in the formation of stud male chemosignals, and the olfactory luteotrophic memory of stud male is further proved to be a short-term one. The precise mechanism involved in accessing and retaining the stud male chemical cues is unclear. In this brief review an attempt has been made to bring out the luteotrophic process of stud male chemosignals, the olfactory pathway and critical period to access the signals. The possible neural mechanism and neural chemistry underlying the formation and recognition of mating male chemical cues are also highlighted.

Characterization of salivary protein during ovulatory phase of menstrual cycle through MALDI-TOF/MS.

Context: Predicting ovulation is the basis on which the fertile period is determined. Nowadays there are many methods available to detect the ovulatory period. Unfortunately, these methods are not always effective for accurate detection of ovulation. Hence, an attempt was made to detect ovulation through single dimension sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein with the help of saliva ferning. Aims: The aim of this study was to determine the association of protein level with endogenous reproductive hormone level across the menstrual cycle. Settings and Design: Salivary protein and its confirmation were evaluated during menstrual cycle followed by SDS-PAGE and Mass spectrometry. Statistical Method Used: The protein content present in saliva throughout menstrual cycle is trail by SPSS statistical software version. Materials and Methods: Salivary proteins were investigated serially during pre-ovulatory, ovulatory and post-ovulatory periods of normal menstrual cycle in eighteen healthy volunteers. The samples were collected in three consecutive menstrual cycles. Salivary protein was estimated and analyzed by single dimension SDS-PAGE. Results: The results revealed significant variations in protein concentrations during the menstrual cycle. Protein levels were maximum during ovulation and minimum during postovulatory phase. Further, single dimension SDS-PAGE analysis showed seven different fractions of proteins is from 14-90 kilo Dalton (kDa) in the three phases of the menstrual cycle. Conclusions: Among the proteins, 48 kDa protein was more predominantly exhibited during ovulatory phase than pre and post-ovulatory phase. The present study indicates that the protein level and the specific protein band (48 kDa) through MALDI-TOF MS analysis might serve as an indicator for ovulation.

Diversity and community structure of butterfly of Arignar Anna Zoological Park, Chennai, Tamil Nadu.

Investigation was carried out on the diversity of butterfly fauna in selected localities of conservation and breeding center of Arignar Anna Zoological Park (AAZP), Chennai, Tamil Nadu. A total of 56 species were recorded, 15 of them belonged to Pieridae, 12 Nymphalidae, 9 Satyridae, 8 Papilionidae, 7 Danaidae, 3 Lycaenidae and 1 species each belonged to the families Acraeidae and Hesperidae. Qualitatively and quantitatively Pieridae family were comparatively dominant than that of other families. The notable addition to the 25 more species listed during this observation were compared to previous field survey. Comparison of butterfly species distribution between the different localities revealed that butterfly species richness was higher at mountain region with 52 species and lowest of 25 species at public visiting areas. Visitor’s activities may be that reason for effects on butterfly distribution and lack of vegetation. Each five endemic and protected species (i.e. endangered) listed under the Wildlife (Protection) Act were highlighted greater conservation importances of the AAZP. It is suggest that butterfly species diversity generally increase with increase in vegetation and declines with the increase in disturbance.

Comparison of physico-chemical parameters and phytoplankton species diversity of two perennial ponds in Sattur area, Tamil Nadu.

Investigations were carried out on the diversity of phytoplankton in relation to physico-chemical parameters with respect to pollution status of two perennial ponds of Sattur area, Tamil Nadu. Fifty species were identified belonging to Chlorophyceae, Bacillariophyceae, Cyanophyceae and Euglenophyceae. High value of physico-chemical parameters and low phytoplankton diversity were recorded in the Chinnapperkovil pond, whereas low value of physico-chemical parameters and high phytoplankton diversity were recorded in the Nallanchettipatti pond. Class Chlorophyceae qualitatively and quantitatively dominated in both the habitats when compared to other taxa. Present study revealed that phytoplankton species richness (R1 and R2) was comparatively higher (R1: 5.43±0.53; R2: 3.00±0.27) in Nallancheettipatti pond. The species diversity was high (H’: 3.08±0.37; N1: 14.05±0.63; N2: 12.55±0.94) in Nallanchettipatti pond compared to Chinnapperkovil pond (H’: 2.25±0.15; N1: 12.59±0.80; N2: 8.54±0.61). Anabaena aequalis, Nitzschia bilobata, Navicula membranacea, Scenedesmus annatus, Pediastrum leonensis, Frustulia rhomboides, Microcystis aeruginosa, Oscillatoria angusta, Closterium acerosum species dominated in Chinnapperkovil pond and Spirogyra maxima, Zygnema caeruteum and Fragilaria oceanica dominated in Nallanchettipatti pond. Abundance of such specific taxa (e.g. Closterium acerosum, C. dianae, C. lineatum, Anabaena aequalis, Oscillatoria angusta and Navicula membranacea) in the Chinnapperkovil and Nallanchettipatti (e.g. Merismopedia glauca and Fragilaria oceanica) ponds suggests that these taxa can be considered as pollution indicators. Phytoplankton species diversity and physico-chemical parameter profiles indicate the Chinnapperkovil pond to be meso-eutrophic whereas the Nallanchettipatti pond is oligo-eutrophic.

Zooplankton diversity and physico-chemical conditions in three perennial ponds of Virudhunagar district, Tamilnadu.

Plankton diversity and physico-chemical parameters are an important criterion for evaluating the suitability of water for irrigation and drinking purposes. In this study, we tried to assess the zooplankton species richness, diversity, and evenness and to predict the state of three perennial ponds according to physico-chemical parameters. A total of 47 taxa were recorded: 24 rotifers, 9 copepods, 8 cladocerans, 4 ostracods and 2 protozoans. More number of zooplankton species were recorded in Chinnapperkovil pond (47 species) followed by Nallanchettipatti (39 species) and Kadabamkulam pond (24 species). Among the rotifers, Branchionus sp. is abundant. Diaphanosoma sp. predominant among the cladocerans. Among copepods, numerical superiority was found in the case of Mesocyclopes sp. Cypris sp. repeated abundance among ostracoda. Present study revealed that zooplankton species richness (R1 and R2) was comparatively higher (R1: 4.39; R2: 2.13) in Chinnapperkovil pond. The species diversity was higher in the Chinnapperkovil pond (H’: 2.53; N1: 15.05; N2: 15.75) as compared to other ponds. The water samples were analyzed for temperature, pH, electrical conductivity, alkalinity, salinity, phosphate, hardness, dissolved oxygen and biological oxygen demand. Higher value of physico-chemical parameters and zooplankton diversity were recorded in Chinnapperkovil pond as compared to other ponds. The zooplankton population shows positive significant correlation with physico-chemical parameters like, temperature, alkalinity, phosphate, hardness and biological oxygen demand, whereas negatively correlated with rainfall and salinity. The study revealed that the presence of certain species like, Monostyla sp., Keratella sp., Lapadella sp., Leydigia sp., Moinodaphnia sp., Diaptomus sp., Diaphanosoma sp., Mesocyclopes sp., Cypris sp. and Brachionus sp. is considered to be biological indicator for eutrophication.

Biochemical evaluation in human saliva with special reference to ovulation detection.

Aim : The aim of the present study was to investigate the level of salivary sialic acids and glycosaminoglycans with reference to salivary hormones during the normal menstrual cycle. Settings and Design: Fifty women volunteers were selected for the present study. Materials and Methods : Saliva was collected from 50 women and ovulation was detected in women with normal menstrual cycles through basal body temperature (BBT), ultrasound and salivary ferning. Samples were divided into five categories, as prepubertal (6-9 years), pre-ovulatory phase (6-12 days), ovulatory phase (13-14 days), postovulatory phase (15-26 days) and menopause (above 45 years). Each sample was subjected to evaluation of the sialic acids and glycosaminoglycans along with salivary hormones. Results : The result revealed that the ovulatory phase has increased sialic acid and glycosaminoglycans during the menstrual cycle when compared with that of the other phases. Consequently, an increased level of hormones such as luteinizing hormone and estrogen during the ovulatory period when compared to that of the pre-ovulatory and postovulatory periods appeared to be noteworthy. Statistically, analysis was performed using one way-ANOVA (LSD; post hoc method) to determine the significance as P < 0.001, 0.01, 0.05 in between the reproductive phases of the menstrual cycle. Conclusion : This study concluded that saliva-specific carbohydrates in the ovulatory saliva make the possibility to develop a biomarker for detection of ovulation by non-invasive methods.

Identification of 2u-globulin and bound volatiles in the Indian common house rat (Rattus rattus)

The 2u-globulin (2u) is a pheromone carrier urinary protein believed to be relevant for sexual communication among rats and is characterized in laboratory rats. In the present study 17 kDa protein and the bound pheromones were characterized in a population of wild-type Indian common house rat (Rattus rattus). The protein was purified by two runs of Sephadex G-50 chromatography and analyzed with SDS-PAGE with MALDI-TOF/MS. The results of MASCOT search identified the protein as an 2u and suggested a role for binding pheromones. To confirm the protein bound volatiles, purified 2u was extracted with dichloromethane and volatile molecules were detected using of gas chromatography linked to mass spectrometry (GC-MS). 1-Chlorodecane was detected as the predominant compound and 2-methyl-N-phenyl-2-propenamide, hexadecane and 2,6,11-trimethyl decane as the minor compounds. The simple method of protein purification and the identification of bound volatiles may help in designing efficient pheromone-based rat traps.

Identification of alpha-2u globulin in the rat preputial gland by MALDI-TOF analysis.

The low molecular mass proteins found in the pheromonal sources such as urine, saliva, glandular secretion etc have been reported as ligand carriers for the processes of chemocommunication in mammals. The preputial gland plays an important role in the production of olfactory signals for pheromonal communication. Thus, in the present study, alpha-2u globulin having molecular mass of 18 kDa has been identified in the preputial gland of Norway rat (Rattus norvegicus) by in-gel trypsin digestion and analyzing the resulting peptides by MALDI-TOF. Since preputial gland is one of the major pheromonal sources in rat, the results suggest that alpha-2u globulin might act as a carrier for hydrophobic odorants of preputial gland.

Evaluation of anticardiolipin antibodies and antiphosphatidylserine antibodies in women with recurrent abortion.

BACKGROUND: Antiphospholipid syndrome (APS) is a major reproductive complication in women, which is characterized by recurrent fetal loss, thrombosis, and thrombocytopenia in association with anticardiolipin antibodies (aCL). AIMS: To analyze the prevalence of aCL and antiphosphatidylserine antibodies (aPS) in relation to pregnancy failures in women with the history of recurrent spontaneous abortion. SETTINGS AND DESIGN: A sequential study of 155 patients, who had three or more recurrent spontaneous abortions, was carried out. METHODS AND MATERIALS: Women with unexplained recurrent pregnancy loss in first trimester were selected for this study. Anticardiolipin antibodies IgG and aPS IgG were detected in the serum by the enzyme linked immunosorbent assay method. STATISTICAL ANALYSIS: Percentage calculation was carried out. Two-tailed t-test was performed to know the significance of aCL and aPS total population. RESULT: The levels of aCL IgG and aPS IgG were detected as 40% (62) and 19% (18), respectively in women with history of recurrent abortion. CONCLUSION: Anticardiolipin antibody is found to be the most important factor for recurrent abortion. In addition, women with negative aCL are having positive for another antiphospholipid antibodies like aPS, which may involve in recurrent abortion.

Identification of glandular (preputial and clitoral) proteins in house rat (Rattus rattus) involved in pheromonal communication.

Proteins (18-20 kDa) belonging to lipocalin family have been reported to act as carriers for ligands binding to pheromones in mouse urine, pig saliva, hamster vaginal fluid and human sweat, that are involved in pheromonal communication. As the preputial gland is a major pheromonal source, the present study was aimed to detect the specific protein bands (around 18-20 kDa) in the preputial and clitoral glands of the house rat, R. rattus. The amount of protein was higher in preputial gland of the male than that of female (clitoral) gland. A 20 kDa protein was noted in male and female glands; however, the intensity of the band was much higher in male than in female. In addition, 70, 60, 35 kDa bands, identified in male preputial gland, were absent in females. The presence of higher concentration of glandular proteins in the male preputial gland suggests that male rats may depend more on these glandular proteins for the maintenance of reproductive and dominance behaviours. The results further suggest that these glandular proteins (20 kDa) may act as a carrier for ligand binding.

Characterization of urinary volatiles in Swiss male mice (Mus musculus): bioassay of identified compounds.

The present study was carried out to investigate the chemical nature of the urine of male mice and to assess its bioactivity. Urine of mature male mice was extracted with dichloromethane (1 : 1 ratio v/v) and analysed by gas-chromatography linked mass-spectrometry (GC-MS). Ten different compounds such as alkanes, alcohols, etc. were detected in the urine. Among the ten, five compounds are specific to males namely, 3-cyclohexene-1-methanol (I), 3-amino-s-triazole (II), 4-ethyl phenol (III), 3-ethyl-2,7-dimethyl octane (IV) and 1-iodoundecane (V). The compound, 4-ethylphenol, has been previously reported in several strains of male mice. Furthermore, the compounds (II) and (IV) are closely similar to 2-sec-butylthiazole and dehydro-exo-brevicomin compounds which have already been reported in male mice. Bioassay revealed that compounds (II), (III) and (IV) were responsible for attracting females and in inducing aggression towards males, as compared to the other compounds i.e. (I) and (V). The results indicate that these three volatiles (II, III and IV) of male mice appear to act as attractant of the opposite sex.

Urinary proteins and pheromonal communication in mammals.

Urinary proteins play a significant role as pheromones and pheromone-binders in mammalian reproduction and social behaviour. The present study was carried out to quantify the urinary proteins in five different mammalian species viz mouse, rat, rabbit, bovine and human. The results revealed that the male rodents excrete large amounts of urinary protein as compared to that of other mammals. In addition, the male mammals excrete a higher quantity of protein than do the females., suggesting the role of androgens in excretion of protein. The presence of higher concentration of urinary proteins in rodents suggests that the rodents depend more on urinary proteins for olfactory/social communication.

Role of male scent glands in improving poison bait acceptance in female rats, Rattus norvegicus.

The poison (Zn3P2) bait mixed with preputial gland and cheek gland extract was highly effective as compared to poison bait mixed without the scent gland extract, in increasing the food consumption and mortality rate in rats. Among these two scent glands, preputial gland extract was more effective than cheek gland extract in increasing the bait acceptance. The scent gland extract mixed with poison bait was capable of improving the poison bait acceptance. A 10% concentration of scent gland extract was more effective than 5% concentration in acceptance of poison bait. The results suggest efficacy of scent gland in improving the poison bait acceptance.

Rat cheek gland compounds: behavioural response to identified compounds.

Cheek gland secretions from sexually mature and reproductively active male and female laboratory rats (Wister strain), Rattus norvegicus albinus were analysed by gas chromatography/mass spectroscopy. Alkanes, aliphatic acids esters and alcohols were identified in the secretions. Cheek gland preparation from males contained predominantly three compounds, viz. di-n-octyl phthalate (I); 1,2-benzene diacarboxylic acid butyl (2-ethyl hexyl) ester (II); and 1,2 benzene dicarboxylic acid (2-methylpropyl) ester (III) whereas in the cheek gland of females two compounds, viz, 1,2-benzene dicarboxylic acid (2-methylpropyl) ester (I); and 2,6,10 dedecatrien-1-ol, 3,7,11-trimethyl-(Z, E) (II) were the major fractions. The identified compounds were tested for odour preferences. Compounds II and III of males and I and II of females attracted both male and female conspecifics. By contrast, compound I from males only attracted females. The level of attraction also varied from compound to compound. The results suggest that cheek gland secretions have pheromonal functions.

Chemistry of clitoral gland secretions of the laboratory rat: assessment of behavioural response to identified compounds.

The present investigations were carried out to find out the chemical nature of clitoral gland extracts and their involvement in reproductive and social behaviour. Homogenates of clitoral glands of mature estrous female rats were extracted with n-hexane and dichloromethane (1 : 1 ratio v/v) and analysed by gas chromatography linked mass spectrometry (GC-MS). Three peaks were found to be in higher concentration, which were identified as 6,11-dihydro-dibenz-b,e-oxepin-11-one (I); 2,6,10-dodecatrien-1-ol-3,7,11-trimethyl(Z) (II); and 1,2-benzene-dicarboxylic acid butyl(2-ethylpropyl) ester (III). Odour preference tests demonstrated that the first compound attracted conspecifics of the opposite sex. By contrast, the second and third compounds were found to attract both sexes. The results conclude that the clitoral gland of laboratory rat contains three major chemical compounds which have a unique function in maintaining social and reproductive status.

Identification of volatile compounds from cheek glands of lesser bandicoot rats and assessment of behavioural response for identified compounds.

Check gland secretions were collected from sexually mature male and female rats to identify the volatile compounds using gas chromatography linked mass spectroscopy with a modified head space technique. Twenty one volatile fractions including alkanes, aliphatic acids, esters and alcohols were found. Three compounds viz. 3-octen-1-ol (E) (I), cyclopentane undecanoic acid (II) and 2,4,6,8 tetramethyl-l-undecane (III) were present in very high concentrations in both male and female rats. In addition, the estrus female contained another compound, L-alanine, 1,1-dimethyl ethyl ester. On the basis of odour preference tests the compounds I, II and III were found to attract both male and female of the homotypic species. In contrast, the fourth compound present in the female attracts the opposite sex. The results suggest that the volatile fractions have unique function and the level of attraction varies from compound to compound.

Influence of the stage of the cycle on olfactory sensitivity in laboratory mice.

Females in estrus showed maximum olfactory sensitivity as judged by their ability in locating the buried bait. The results suggest that olfactory sensitivity in females varies during the stages of the estrous cycle. The findings further indicate that gonadal steroids play an important role in the expression of olfactory sensitivity in females.

Nutritional stress-induced implantation failure in laboratory mice: inhibition by continuous illumination.

The implantation failure in newly inseminated mice induced by food deprivation for 48 hr, beginning at 0900 hrs on day 4 post coitum, was prevented by simultaneous exposure to light continuously for 48 or 36 hr. Food-deprived females that were exposed to continuous light for 36 hr showed a significant increase in fetal resorption as compared with food-deprived females exposed to continuous light for 48 hr. Since failure of hypophysial prolactin release appears to be the primary endocrine cause of the inanition-induced implantation failure, the results suggest that exposure to continuous light protects implantation in food-deprived females by stimulating luteotrophic activity.