Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Reproductive Biology
journal homepage: www.elsevier.com/locate/repbio
Original article
Center for Pheromone Technology, Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli, 620024, India
Research Institute in Semiochemistry and Applied Ethology, Quartier Salignan, 84 400 Apt, France
c
Centre for Animal Research, Training and Services (CAReTS), Central Inter-Disciplinary Research Facility (CIDRF), Mahatma Gandhi Medical College and
Research Institute (MGMC-RI) Campus, Puducherry, 607403, India
d
Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 143-701, Republic of Korea
e
Translational Neuroscience Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 636921, Singapore
f
National Centre for Alternatives to Animal Experiments, Bharathidasan University, Tiruchirappalli, 620024, India
g
Department of Food Science and Nutrition, College of Food Science and Agriculture, King Saud University, Riyadh, Saudi Arabia
b
A R T I C L E I N F O
Article history:
Received 17 May 2016
Received in revised form 19 October 2016
Accepted 19 October 2016
Available online xxx
Keywords:
Functional annotation
Biomarker
SDS-PAGE
Protein
Cystatin-S
A B S T R A C T
Human saliva contains numerous molecules that play a variety of roles. Among them there are proteins
which serve as biomarkers of various physiological and/or pathological conditions. Compared to other
body uids, saliva is the most convenient material for investigations, and especially for monitoring the
disease conditions. Presently, there is an increasing need to develop a noninvasive method to identify the
time of ovulation in humans to ensure successful fertilization, and for evolving strategies for family
planning. The present investigation has been an attempt to identify one or more proteins in the human
saliva that would be an indicator(s) of ovulation. SDS-PAGE of salivary proteins showed seven prominent
bands during the different phases of the menstrual cycle. Particularly, the 14.5 kDa band was highly
expressed during the ovulatory phase. Eleven proteins were identied in this band of which ten were
highly specic to the ovulatory phase. Among those proteins the intense expression of Cystatin-S was
validated using immunoblot analysis (p < 0.05). The functional annotation of salivary proteins revealed a
high percentage of proteins that engage in binding and regulatory activities. The present results indicate
that salivary proteins, particularly those present during the ovulatory phase, might be used as biomarkers
for impending ovulation.
2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of
Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved.
1. Introduction
Human saliva contains major, minor and gingival crevicular
secretions from parotid, submandibular and sub-lingual glands,
which play pivotal roles in digestion of food and maintaining the
oral health [1,2]. Saliva is an excellent biological uid that is useful
for noninvasive exploration of the human diseases and physiological conditions [3]. It contains various biomolecules such as
proteins, enzymes and hormones [4,5]. However, the concentration of biomolecules in saliva is generally only one-tenth of that in
the blood [6]. More than two thousand proteins and peptides have
been identied [7] in different secretions of the major salivary
glands [8]. The salivary proteins facilitate bacterial agglutination
[9], digestion of food, antimicrobial activity, lubrication and
cleaning [10].
The salivary gland secretion is regulated by the autonomic
nervous system. The sympathetic and parasympathetic nervous
systems effectively regulate ow rate and composition of saliva.
The parasympathetic system facilitates secretion of a high volume
of saliva containing fewer proteins, whereas the sympathetic
http://dx.doi.org/10.1016/j.repbio.2016.10.005
1642-431X/ 2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by
Elsevier Sp. z o.o. All rights reserved.
Please cite this article in press as: G. Saibaba, et al., Proteomic analysis of human saliva: An approach to nd the marker protein for ovulation,
Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2016.10.005
G Model
REPBIO 208 No. of Pages 8
system has the opposite effect [11]. The enzyme amylase (ptyalin)
and mucins are the major constituents in salivary proteome [12].
Saliva contains immunoglobulins also, and 60% of the total salivary
immunoglobulins is immunoglobulin A (IgA) [13]. Albumin has
also been detected as present in saliva but its concentration varies
from person to person [14]. The human salivary proteins which are
of low molecular weight, such as histatin and proline-rich proteins,
(PRPs) contribute greatly to the oral health [15].
Ovulation is a biological process wherein the mature ovarian
follicle ruptures so as to discharge the ovum, and this happens
under the inuence of luteinizing hormone (LH) surge. The LH
surge triggers a series of proteolytic processes which control
ovulation [16]. Saliva contains a number of hormones. The levels of
estrogen and progesterone in the saliva of premenopausal women
vary in relation to the phases of the menstrual cycle, and the
uctuation correlates with that in blood serum [17]. Similarly, the
salivary testosterone and cortisol levels have been evaluated to
diagnose hypogonadism in males by adopting liquid chromatography-tandem mass spectrometry (LCMS/MS). Compared to other
hormones in human saliva, cortisol exhibits a noticeable diurnal
variation [18].
Over the past decade, proteomics have been considered as one
of the best approaches for identication of biomarkers for various
diseases [19]. Salivary proteins have been identied as biomarkers
for various disease conditions such as Sjogrens syndrome [20],
lung cancer [21], oral cancer [22], a number of systemic diseases
[23], HIV infection [24], dental pellicle development [25], and
hyperglycemia [26]. However, no salivary protein marker for
monitoring the time of ovulation in women has been reported to
date. Although crystallization of saliva is useful in identication of
the fertile period in women [27], this method does not ensure
sufcient sensitivity and specicity. Since no simple and reliable
technique or biomarker is yet available for detection of ovulation in
women, a rapid protein-based detection kit, similar to the
noninvasive pregnancy detection kit that is based on the presence
of hCG (human chorionic gonadotropin) in urine [28], is very much
needed. Perhaps, one or more salivary proteins would serve as a
potential noninvasive biomarker(s) to predict ovulation [29].
Prediction of ovulation in women will be of application in assisted
reproductive technologies (ART), in vitro fertilization (IVF), and
natural family planning. Therefore, an attempt has been made in
this study to investigate the prole of salivary proteins during the
various phases of the menstrual cycle to identify the proteins
which are specically present during the ovulation phase, adopting
high resolution liquid chromatography tandem mass spectrometry
(LCMS/MS).
2. Materials and methods
2.1. Chemicals
The chemicals and reagents used in this study were obtained
from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
2.2. Ethics statement
Sample collection from women volunteers and all experimental
protocols were approved by the Institutional Ethical Committee
(IEC) (Approval No: DM/2014/101/38), Bharathidasan University,
Tiruchirappalli, India. All procedures were carried out in accordance with the approved guidelines. Written consent was obtained
from the volunteers who participated in the study.
Please cite this article in press as: G. Saibaba, et al., Proteomic analysis of human saliva: An approach to nd the marker protein for ovulation,
Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2016.10.005
G Model
REPBIO 208 No. of Pages 8
Antimicrobial activity of the whole saliva was assessed by welldiffusion method according to the National Committee for Clinical
Laboratory Standards (NCCLS). Mueller-Hinton agar (MHA) plates
were inoculated with 12 h old broth cultures of the test organisms
to create conuent lawns of bacterial growth. The antibacterial
effect of saliva was inferred from the colony-free zone around the
well into which the saliva was introduced. The agar plates were
incubated at 37 C for 24 h and the inhibitory pattern was
determined by measuring the diameter of the zone of inhibition
around the well (in mm). The experiments were repeated thrice
and the average zone of inhibition was calculated.
Fig. 1. Salivary estradiol levels during menstrual cycle. The daily pattern of estradiol
level was recorded in the saliva sample of the subject. The estradiol level reached
the peak around day 13 or 14 of menstrual cycle. Data are presented as mean SD.
Please cite this article in press as: G. Saibaba, et al., Proteomic analysis of human saliva: An approach to nd the marker protein for ovulation,
Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2016.10.005
G Model
REPBIO 208 No. of Pages 8
Table 1
Concentrations of salivary proteins during the different phases of menstrual cycle.
Details
Pre-Ovulation Ovulation
2.49 0.39*
Post-Ovulation
1.50 0.28**,a
The values are expressed as mean SD. The mean difference is signicant at
p < 0.05.
*
p < 0.05, Pre-ovulation v Ovulation.
**
p < 0.05, Ovulation v Post-ovulation.
a
p < 0.05, Pre-ovulation v Post-ovulation.
Fig. 2. SDS-PAGE of whole salivary proteins. The salivary proteins were separated
by 12% SDS-PAGE. Lane-M, medium range molecular markers. The concentration of
the protein samples in the different lanes was the same. PreO Pre-ovulation phase,
O Ovulation phase, PostO Post-ovulation phase.
Table 2
List of proteins identied in 14.5 kDa band of ovulatory phase.
Swiss-Prot acc. no.a
Name
Functionb
pIc
MWc
P01036
P01037
P09228
P04080
P12273
P01040
P61626
P01034
P10599
Cystatin-S
Cystatin-SN
Cystatin-SA
Cystatin-B
Prolactin-inducible protein
Cystatin-A
Lysozyme C
Cystatin-C
Thioredoxin
4.83
6.92
4.85
6.96
5.40
5.38
9.38
8.75
4.82
14.18
14.31
14.35
11.13
13.52
11.00
14.70
13.34
11.60
141
141
141
98
146
98
P61769
P51649
Q9H2U9
Beta-2-microglobulin
Succinate-semialdehyde dehydrogenase
Disintegrin and metalloproteinase domain-containing
protein 7
Coiled-coil domain-containing protein 171
Uncharacterized protein KIAA0232
Strong inhibitor
Proteinase inhibitor
Thiol proteinase inhibitor
Intracellular thiol proteinase inhibitor
Protein binding
Intracellular thiol proteinase inhibitor
Bacteriolytic function
Inhibitor of cysteine proteinases
Expression of interleukin-2 receptor
TAC
Antigen presentation
Catalysis
Role in reproduction
1
1
7
Unknown
Nucleotide binding
8
1
Q6TFL3
Q92628
a
b
c
146
13
11
7
4
2
2
3
3
1
Proteins having at least one identied peptides in ovulation period saliva are listed with their Swiss-Prot/TrEmbl accession numbers and length.
Functions were retrieved using the STRAP online database bioinformatics resource [35].
Theoretical pIs (c) and monoisotopic molecular weights (d) were calculated using the Swiss-Prot website [61].
Please cite this article in press as: G. Saibaba, et al., Proteomic analysis of human saliva: An approach to nd the marker protein for ovulation,
Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2016.10.005
G Model
REPBIO 208 No. of Pages 8
Fig. 3. Western blot analysis of salivary protein. A) Western blot of Cystain-S in saliva during different phases of menstrual cycle. B) The bar diagram represents relative
abundance of Cystatin-S during the three phases, of the menstrual cycle normalized with the expression of the housekeeping gene b-actin. Equal amount of protein was
loaded to each lane and data are presented as mean SD. *p < 0.05 as compared with pre- and post- ovulation phases. PreO- Pre-Ovulation, O- Ovulation, PostO- PostOvulation.
Fig. 4. Antimicrobial activity of saliva during the three phases of menstrual cycle.
The antibacterial activity of saliva was tested against test strains by well diffusion
method. The ovulation phase saliva shows highest activity (Inhibition zone- 6 and
5 mm respectively) towards K. pneumoniae and E. coli.
Table 3
Functional annotation of salivary proteins during the ovulatory phase.
Biological process
Cellular location
Molecular function
Process
Percent
Location
Percent
Function
Percent
Regulation
Immune system process
Cellular process
Interaction with cells and organisms
Localization
Metabolic process
Response to stimulus
Developmental process
Others
41.31%
17.13%
16.2%,
15.11%
14.11%
10.8%
7.5%
6.5%
6.5%
Extracellular
Plasma membrane
Cytoplasm
Nucleus
Endosome
Macromolecular complex
Cytoskeleton
Mitochondria
ER
Cell surface
Chromosome
Other intracellular organelles
Others
39.5%
16.14%
10.9%
10.9%
5.4%
3.3%
3.3%
3.3%
3.3%
1.1%
1.1%
10.9%
8.7%
Binding
Catalytic activity
Enzyme regulator activity
Structural molecule activity
Antioxidant activity
Others
46.60%
17.22%
10.13%
1.1%
1.1%
2.3%
Please cite this article in press as: G. Saibaba, et al., Proteomic analysis of human saliva: An approach to nd the marker protein for ovulation,
Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2016.10.005
G Model
REPBIO 208 No. of Pages 8
Please cite this article in press as: G. Saibaba, et al., Proteomic analysis of human saliva: An approach to nd the marker protein for ovulation,
Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2016.10.005
G Model
REPBIO 208 No. of Pages 8
[20] Ryu OH, Atkinson JC, Hoehn GT, Illei GG, Hart TC. Identication of parotid
salivary biomarkers in Sjogrens syndrome by surface-enhanced laser
desorption/ionization time-of-ight mass spectrometry and two-dimensional
difference gel electrophoresis. Rheumatology (Oxford) 2006;45:107786.
[21] Xiao H, Zhang L, Zhou H, Lee JM, Garon EB, Wong DT. Proteomic analysis of
human saliva from lung cancer patients using two dimensional difference gel
electrophoresis and mass spectrometry. Mol Cell Proteom 2012;11:112.
[22] Hu S, Wang J, Meijer J, Ieong S, Xie Y, Yu T, et al. Salivary proteomic and genomic
biomarkers for primary Sjogrens syndrome. Arthritis Rheum 2007;56:3588
600.
[23] Xiao H, Wong DT. Proteomics and its applications for biomarker discovery in
human saliva. Bioinformation 2010;5:2946.
[24] Zhanga N, Zhang Z, Feng S, Wang Q, Malamud D, Deng H. Quantitative analysis
of differentially expressed saliva proteins in human immunodeciency virus
type 1 (HIV-1) infected individuals. Anal Chim Acta 2013;774:616.
[25] Gibbins HL, Proctor GB, Yakubov GE, Wilson S, Carpenter GH. Concentration of
salivary protective proteins within the bound oral mucosal pellicle. Oral Dis
2014;20:70713.
[26] Bencharit S, Baxter SS, Carlson J, Byrd WC, Mayo MV, Border MB, et al. Salivary
proteins associated with hyperglycemia in diabetes: a proteomic analysis. Mol
BioSyst 2013;9:278597.
[27] Braat DD, Smeenk JM, Manger AP, Thomas CM, Veersema S, Merkus JM. Saliva
test as ovulation predictor. Lancet 1998;352:12834.
[28] Evans HM. Simpson ME. AschheimZondek test for pregnancy-its present
status. Cal West Med 1930;32:1458.
[29] Saibaba G, Archunan G. Does salivary protein(s) act an ovulation indicator for
women? A hypothesis. Med Hypotheses 2015;84:1046.
[30] Navazesh M. Methods for collecting saliva. Ann N Y Acad Sci 1993;20:727.
[31] Alagendran S, Saibaba G, Muthukumar S, Rajkumar R, Guzman RG, Archunan
G. Characterization of salivary protein during ovulatory phase of menstrual
cycle through MALDI-TOF/MS. Ind J Dent Res 2013;24:15763.
[32] Bradford MM. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal
Biochem 1976;72:24854.
[33] Muthukumar S, Rajesh D, Saibaba G, Alagesan A, Rengarajan RL, Archunan G.
Urinary lipocalin protein in a female rodent with correlation to phases in the
estrous cycle: an experimental study accompanied by in silico analysis. PLoS
One 2013;8:e71357.
[34] Vizcano JA, Deutsch EW, Wang R, Csordas A, Reisinger F, Ros D, et al.
ProteomeXchange provides globally coordinated proteomics data submission
and dissemination. Nat. Biotechnol 2014;30:2236.
[35] Bhatia VN, Perlman DH, Costello CE, McComb ME. Software tool for
researching annotations of proteins: open-source protein annotation
software with data visualization. Anal Chem 2009;81:981923.
[36] Ulcova-Gallova Z. Immunological and physicochemical properties of cervical
ovulatory mucus. J Reprod Immunol 2010;86:11521.
[37] Fernando RS, Regas J, Betz G. Physiological mechanisms associated with
ovulation prediction using the CUE Ovulation Predictor. Hum Reprod
1988;3:4137.
[38] Zondek B. Arborization of cervical and nasal mucus and saliva. Obstet Gynecol
1959;13:47781.
[39] Muthukumar S, Rajkumar R, Rajesh D, Saibaba G, Liao CC, Archunan G, et al.
Exploration of salivary proteins in buffalo: an approach to nd marker proteins
for estrus. FASEB J 2014;28:110.
[40] Rajkumar R, Karthikeyan K, Archunan G, Huang PH, Chen YW, Ng WV, et al.
Using mass spectrometry to detect buffalo salivary odorant-binding protein
and its post-translational modications. Rapid Commun Mass Spectrom
2010;24:324854.
[41] Andersch-Bjorkman Y, Thomsson KA, Holmn Larsson JM, Ekerhovd E,
Hansson GC. Large scale identication of proteins, mucins, and their Oglycosylation in the endocervical mucus during the menstrual cycle. Mol Cell
Proteom 2007;6:70816.
[42] de Almeida PDV, Grgio AM, Machado MA, de Lima AA, Azevedo LR. Saliva
composition and functions: a comprehensive review. J Contemp Dent Pract
2008;9:110.
[43] Kutteh WH, Prince SJ, Hammond KR, Kutteh CC, Mestecky J. Variations in
immunoglobulins and IgA subclasses of human uterine cervical secretions
around the time of ovulation. Clin Exp Immunol 1996;104:53842.
[44] Schenkels LC, Veerman EC, Nieuw Amerongen AV. Biochemical composition of
human saliva in relation to other mucosal uids. Crit Rev Oral Biol Med
1995;6:16175.
[45] Amerongen AV, Veerman EC. Saliva: the defender of the oral cavity. Oral Dis
2002;8:1222.
[46] Achiraman S, Archunan G. Urinay proteins and pheromonal communication in
mammals. Ind J Exp Biol 2002;40:10778.
[47] Chatterjee S, Damle SG, Sharma AK. Salivary heat shock proteins and their
interactions with oral microenvironment. Inamm Cell Signal 2014;1:7683.
[48] Vanmuylder N, Evrard L, Daelemans P, Van Reck J, Dourov N. Expression of heat
shock proteins in salivary gland tumors Immunohistochemical study of HSP27,
HSP70, HSP90, and HSP110: apropos of 50 cases. Ann Pathol 2000;20:1905.
[49] Imamura Y, Wang P. Salivary histatin 3 inhibits heat shock cognate protein 70mediated inammatory cytokine production through toll-like receptors in
human gingival broblasts. J Inamm 2014;11:4.
[50] Espey L. Ovulation as an inammatory reaction: a hypothesis. Biol Reprod
1980;22:73106.
Please cite this article in press as: G. Saibaba, et al., Proteomic analysis of human saliva: An approach to nd the marker protein for ovulation,
Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2016.10.005
G Model
REPBIO 208 No. of Pages 8
[56] Dickinson DP. Salivary (SD-type) cystatins: over one billion years in the
making-but to what purpose? Crit Rev Oral Biol Med 2002;13:485508.
[57] pela Magister S, Kos J. Cystatins in immune system. J Cancer 2013;4:4556.
[58] Mirtti T, Alanen K, Kallajoki M, Rinne A, Sderstrm KO. Expression of
cystatins, high molecular weight cytokeratin, and proliferation markers in
prostatic adenocarcinoma and hyperplasia. Prostate 2003;54:2908.
[59] Jiborn T, Abrahamson M, Wallin H, Malm J, Lundwall A, Gadaleanu V, et al.
Cystatin C is highly expressed in the human male reproductive system. J
Androl 2004;25:56472.
[60] Alagendran S, Archunan G, Bonnila Armando EO, Guzman RG. Evaluation of
salivary electrolytes during normal menstrual cycle with special reference to
ovulation. Am J Appl Sci 2010;7:106672.
[61] Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A. ExPASy: the
proteomics server for in-depth protein knowledge and analysis. Nucleic Acids
Res 2003;31:37848.
Please cite this article in press as: G. Saibaba, et al., Proteomic analysis of human saliva: An approach to nd the marker protein for ovulation,
Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2016.10.005