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The effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs
Theriogenology 131 (2019) 41e46
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The effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs Marek Szczubiał a, *, Jacek Wawrzykowski b, Roman Da˛browski a, Mariola Bochniarz a, Piotr Brodzki a, Marta Kankofer b a b
Department and Clinic of Animal Reproduction, Faculty of Veterinary Medicine, University of Life Sciences, Głeboka 30, 20-612, Lublin, Poland Department of Animal Biochemistry, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12, 20-033, Lublin, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2019 Received in revised form 20 March 2019 Accepted 24 March 2019 Available online 25 March 2019
The main aim of this study was to investigate the effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs, using western blotting with selected lectins (Sambucus nigra agglutinin - SNA and Maackia amurensis agglutinin e MAL II). In addition protein pattern of examined tissues was also evaluated. The study was performed on 10 female dogs undergoing ovariohysterectomy because of pyometra and 10 clinically healthy female dogs, undergoing elective spaying (ovariohysterectomy). Uterine tissue samples of 1 cm2 were taken from the middle region of each uterine horn in both group of animals immediately after ovariohysterectomy. Tissue samples were homogenized and analysed by sodium dodecyl sulphateepolyacrylamide gel electrophoresis (SDS-PAGE) and western blotting with SNA and MAL II. SDS-PAGE analysis showed differences between pyometra samples and controls in the amount of obtained protein fractions and the protein content in the individual fractions. Five protein (with a molecular weight of 193.78 kDa, 103.18 kDa, 77.67 kDa, 70.39 kDa, and 53.00 kDa) were found only in the pyometra samples. The remaining fractions differed in intensity of staining, which indicated differ abundance of a given protein. The results of western blotting with SNA and MAL II demonstrated that the pattern obtained from densitometric analysis differs between adequate healthy and pyometra samples with regard to the amount of protein fraction obtained as well as the intensity of staining of particular fraction. The pyometra tissues contained seven SNA-binding proteins (with a molecular weight 189.94 kDa, 165.51 kDa, 100.94 kDa, 59.42 KDa, 41.32 kDa, 35.16 kDa, and 32.6 kDa) that were not in the healthy tissues. Of the nine remaining fractions, six showed significantly higher (P < 0.05) intensity of staining in the healthy uterine tissues. In turn, the MAL II-binding protein with a molecular weight 75.85 kDa, 51.12 kDa, and 49.98 kDa were found only in the pyometra samples. Of the 28 remaining fractions, ten demonstrated significantly higher (P < 0.05), and five fractions had significantly lower (P < 0.05) intensity of staining in the pyometra tissues. The results obtained indicate that proteins in uterine tissues from female dogs with pyometra are differently glycosylated compared to normal uterine tissues. These findings provide the basis for further studies of the possible role of glycosylation in the pathogenesis of canine pyometra. © 2019 Elsevier Inc. All rights reserved.
Keywords: SNA MAL II Lectin blotting Pyometra Uterus Female dogs
1. Introduction Pyometra is a common, life threatening uterine disease in intact dogs [1,2]. The disease occurs mainly in geriatric female dogs in the lutheal stage of the oestrus cycle [3,4]. The pathogenesis of pyometra has not been completely established, however, it is
* Corresponding author. E-mail address: [email protected] (M. Szczubiał). https://doi.org/10.1016/j.theriogenology.2019.03.020 0093-691X/© 2019 Elsevier Inc. All rights reserved.
commonly accepted that disease results from bacterial interaction with an endometrium that has undergone pathological changes caused by progesterone and estrogens influence [5e7]. Glycosylation is the most common post-translational modification of proteins which produces different types of glycoconjugates that are typically attached to cellular proteins and lipids [8]. Such post-translational changes can affect many cellular processes, such as cell-cell and cell-matrix recognition, adhesion, motility, control of membrane permeability, and molecular recognition [9,10].
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Glycosylation can occur spontaneously but can also be the result of pathological processes. To analyze glycosylation in normal and pathologically changed tissues many different techniques using lectins, including western blotting, can be used [11e14]. Lectins are glycan binding proteins that are typically, highly selective for specific glycan structures and for that reason are useful in the study of glycan variation [11]. It is known that the mammalian uterus is highly enriched in glycoconjugates that are associated with the apical surfaces of epithelial cells and the secretions released by both epithelium and stromal cells [15]. These glycoconjugates interact primarily with sperm, the implanting embryo, the fetus, and any pathogens that gain entry into the uterus. According to many studies, mainly from human medicine, various pathological processes related with reproduction, such as ovarian and cervical cancer, endometriosis, endometrial degeneration, different hypertensive disorders of pregnancy as well as preterm birth and unexplained infertility, are associated with alternations in glycosylation patterns [12,15e20]. Although many different lectin binding studies have been performed on tissue samples from the human uterus there are very few studies concerning uterine tissues from female dogs [21,22]. Thus, the main aim of this study was to investigate the effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs, using western blotting with selected lectins (SNA and MAL II). In addition protein pattern of examined tissues was also evaluated. 2. Material and methods 2.1. Animals The study was performed in accordance with national animal protection regulations (Animal Experimentation Act dated 15 January 2015) which are in agreement with European legislation about ethics in animal experiments. The study was performed on 20 female dogs of various breeds and mixed-breed undergoing ovariohysterectomy at the Department of Animal Reproduction, Faculty of Veterinary Medicine in Lublin. The first group involved 10 clinically healthy female dogs, aged 2e4 years, weighted 10e17 kg, between 3 and 6 weeks after oestrus, admitted to clinic for elective spaying (ovariohysterectomy) on the owners request. These dogs were classified as healthy after complete physical examination, haematology, biochemistry, and ultrasonography. The second group included 10 female dogs aged 6e12 years, weighted 12e24 kg, between 4 and 7 weeks after oestrus, undergoing ovariohysterectomy because of pyometra. These dogs were diagnosed with pyometra after medical history, physical examination, haematology, biochemistry and ultrasonography. In most cases of dogs with pyometra clinical examination revealed pyrexia, polydipsia, polyuria, anorexia, apathy, and vaginal discharge [1]. Findings on abdominal ultrasound in all affected dogs were consistent with pyometra (an enlarged uterus filled with anechoic to hypoechoic fluid with a thickened wall) [23]. Dogs with some other reproductive or other abdominal changes observed in the abdominal ultrasonography were excluded from the study. To confirm the same phase of oestrus cycle (dioestrus) in both group of dogs vaginal cytology and serum progesterone concentration were performed using commercial ELISA kit (MyBioSource, Ins., San Diego, USA). All dogs were subjected to routine ovariohysterectomy. Normal uterus and pyometra cases were confirmed by postoperative histopathological examination of the uterus by routine histopathological examination. In all cases of pyometra samples of uterine pus were collected
directly from the infected uterine horns for bacterial identification. 2.2. Tissue collection Uterine tissue samples of 1 cm2 were taken from the middle region of each uterine horn in both group of animals immediately after ovariohysterectomy. Luminal exude was excluded from endometrial samples. Tissue samples were homogenized in phosphate buffer (0.1 mol/L, pH 7.0) by use of Ultra Turrax (Ikawerk, Janke, Kunkel, Staufen, Germany), centrifuged at 4 C for 20 min at 6500 g and frozen at 76 C for further analysis. 2.3. Electrophoresis Uterine homogenates were separated by sodium dodecyl sulphateepolyacrylamide gel electrophoresis (SDS-PAGE) [24] on 16.5% polyacrylamide gels (7 5 cm, 1.5 mm), using Mini-PROTEAN Tetra cell system (Bio-Rad, Warszawa, Poland). The amount of protein loaded per each well was the same and amounted 4 mg. The running buffer was 0.025 M Tris/glycine, pH 8.3. Constant voltage of 200 V was used. After electrophoresis, control gels were stained for protein with Roti-Blue colloidal Coomassie brilliant staining (Roti, Karlsruhe, Germany). 2.4. Western blotting Protein fractions were transferred from SDS-PAGE to ImmunBlot PVDF membranes (0.2 mm, polyvinylidene difluoride; BioRad) in accordance with procedure described by Towbin et al. [25] (Criterion blotter (Bio-Rad, Warszawa, Poland), during 2 h at 400 V in 4 C). To eliminate non-specific immune response, membranes were incubated for 1 h in 15 ml 0.05% Tween 20 in R.T.U. animal free block solution (SP-5035, Vector Laboratories, Burlingame, CA, USA). Biotinylated lectins were used as antibodies for appropriate sugar moieties in uterine proteins. Overnight incubation at 4 C with biotinylated Maackia Amurensis lectin II, MAL II (B1265, Vector Laboratories) for glycans that bind sialic acid in a 2,3linkage (1 mg/mL in 0,05% PBST) was performed and respectively overnight incubation 4 C with biotinylated Sambucus Nigra lectin, SNA (B-1305, Vector Laboratories) for glycans that bind sialic acid in a 2,6-linkage (1 mg/mL in 0,05% PBST). Complexes of biotinylated lectins with appropriate glycoproteins were visualized by staining with streptavidin-based conjugated with alkaline phosphatase with the use of reagent kit (VECTASTAIN ABC-AP Staining KIT, AK-5000 and BCIP/NBT Alkaline Phosphatase (AP) Substrate Kit, SK-5400, Vector Laboratories) in accordance with the manufacturer’s procedure. Dark fractions located at selected molecular weight positions were considered as positive reactions. As a positive and negative control, horseradish peroxidase (Peroxidase from horseradish, 77332, Sigma) and trypsin inhibitor (Trypsin inhibitor from Glycine max (soybean), T9003, Sigma) were used on each gel respectively. The membranes were scanned on an imaging densitometer GS710 (Bio-Rad, Warszawa, Poland), and the molecular weight and relative quantities were estimated with the Bio-Rad Quantity One 4.1 software (Bio-Rad). Protein content in the samples was determined according to the method based on the biuret reaction using a commercial colorimetric kit (Cormay, Lublin, Poland) [26]. 2.5. Statistical analysis The results were presented as means ± SD and analysed using the computer program STATISTICA version 10.0 (Statsoft, USA).
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After testing for normal distribution (the Shapiro-Wilk/ Kolmogorov-Smirnov tests), the Student’s t-test was used to compare the results of densitometric analysis between the pyometra and control groups. Differences were considered significant at P < 0.05. 3. Results Microbiological examination identified Escherichia coli in the samples of uterine pus from all female dogs with pyometra. To compare binding patterns of SNA and MAL II in uterus from healthy female dogs and uterus from female dogs with pyometra, 10 individual homogenates of normal uterine tissues and 10 homogenates of diseased uterine tissues were examined by western blotting using these two lectins. Fig. 1 shows the banding patterns of proteins after sodium dodecyl sulphate epolyacrylamide gel electrophoresis (SDS-PAGE) and after western blotting with SNA and MAL II in tissue homogenates from uterus of healthy female dogs and uterus from dogs with pyometra. Densitometric analysis of SDS-PAGE gels showed that the protein composition of the uterine tissues from dogs with pyometra differed from that of the healthy uterus. This analysis demonstrated differences in the amount of protein fractions obtained and the protein content in the individual fractions as expressed by alterations in staining intensity of fraction. The protein with a molecular weight of 193.78 kDa as well as the proteins with molecular weights of 103.18 kDa, 77.67 kDa, 70.39 kDa, and 53.00 kDa were found only in the uterine samples from dogs with pyometra (Fig. 1 e SDS-PAGE separation, Table 1). The remaining fractions differed in intensity of staining, which indicated different abundance of a given protein. In case of proteins with molecular weights of 117.77 kDa, 98.19 kDa, 89.16 kDa, 75.36 kDa, 67.07 kDa, 42.23 kDa, 35.37 kDa, 33.35 kDa, 29.97 kDa and 25.59 kDa differences in intensity of staining reached statistical significance (P < 0.05) (Fig. 1 e SDS-PAGE separation, Table 1). Results of western blotting with SNA and MAL II clearly demonstrated that the pattern obtained from densitometric analysis differed between adequate healthy and pyometra samples with regard to the number of protein fractions obtained as well as the intensity of staining of particular fraction. Generally, the homogenates from dogs with pyometra had markedly increased reactivity to SNA. The pyometra tissues contained seven SNA-binding proteins (with molecular weights of 189.94 kDa, 165.51 kDa, 100.94 kDa, 59.42 KDa, 41.32 kDa, 35.16 kDa, and 32.6 kDa) that were not in the healthy tissues (Fig. 1 e SNA e A, Table 2). Of the nine remaining fractions, five (with molecular weights of 147.92 kDa, 92.14 kDa, 85.20 kDa, 74.91 kDa, and 54.53 kDa) showed significantly higher (P < 0.05) intensity of staining in healthy uterine tissues than in compared pyometra cases. The fraction with a molecular weight 49.77 kDa intensity of staining was significantly higher (P < 0.05) in pyometra samples (Fig. 1 e SNA e B, Table 2). Also proteins in the pyometra samples tended to bind more MAL II and a higher intensity of band staining was observed in these samples. The MAL II-binding proteins with molecular weights of 75.85 kDa, 51.12 kDa, and 49.98 kDa were found only in the
Fig. 1. The patterns of electrophoretic and western blotting separations from uterine samples of pyometra and healthy bitches. Upper pattern e electrophoretic line and its densitometric analysis from SDS-PAGE separation; middle pattern e western blotting pattern and its densitometric analysis from SNA determinations; bottom pattern western blotting pattern and its densitometric analysis from MALL II determinations. (A) e pyometra samples, (B) e control samples. Bottom part of each picture represents example of most representative paths while upper part shows mean results of densitometric analysis of all adequate paths and fractions.
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Table 1 The results of densitometric analysis of SDS-PAGE gels. Molecular weight (kDa)
193.78 165.64 137.31 128.25 117.77 106.02 103.18 98.19 94.20 89.16 77.67 75.36 70.91 70.39 67.07 58.52 53.00 49.75 42.23 39.23 37.13 35.37 33.35 29.97 25.59 a
Table 3 The results of densitometric analysis of MALL II blottings.
Densitometric analysis (arbitrary units) Pyometra group (n ¼ 10)
Control (n ¼ 10)
1.09 ± 0.37 2.14 ± 1.02 0.93 ± 0.47 1.26 ± 0.41 0.95 ± 0.27 1.73 ± 0.72 2.82 ± 0.29 1.27 ± 0.29 1.28 ± 0.70 4.41 ± 1.52 5.10 ± 3.10 0.80 ± 0.33 15.15 ± 9.96 0.92 ± 0.25 19.21 ± 2.93 2.59 ± 0.95 1.41 ± 0.41 7.16 ± 2.06 11.04 ± 1.81 3.23 ± 0.73 2.21 ± 0.81 3.97 ± 1.11 6.51 ± 1.55 2.45 ± 0.72 8.29 ± 6.98
e 1.54 ± 0.36 0.78 ± 0.22 0.98 ± 0.62 0.57 ± 0.15a 1.67 ± 0.37 e 0.48 ± 0.06a 1.60 ± 0.40 1.11 ± 0.08a e 6.50 ± 1.02a e e 28.29 ± 3.52a 2.20 ± 0.41 e 6.06 ± 1.83 14.56 ± 1.10a 3.28 ± 0.37 2.68 ± 0.73 2.58 ± 0.90a 3.57 ± 0.86a 1.02 ± 0.33a 4.26 ± 2.06a
Statistically significant difference at P < 0.05.
Table 2 The results of densitometric analysis of SNA blottings. Molecular weight (kDa)
189.94 165.51 147.92 117.19 100.94 92.14 85.20 74.91 65.29 59.42 54.53 49.77 43.26 41.32 35.16 32.60 a
Densitometric analysis (arbitrary units) Pyometra group (n ¼ 10)
Control (n ¼ 10)
13.4 ± 9.84 6.40 ± 2.64 9.03 ± 2.71 23.36 ± 3.85 19.15 ± 6.27 2.03 ± 1.85 6.98 ± 3.63 2.08 ± 0.27 11.85 ± 6.31 1.66 ± 1.26 0.55 ± 0.07 4.07 ± 1.39 2.37 ± 1.53 4.65 ± 1.98 2.55 ± 0.27 1.66 ± 1.51
e e 20.11 ± 6.93a 19.55 ± 8.71 e 16.77 ± 2.48a 13.98 ± 5.97a 4.57 ± 0.81a 8.31 ± 1.33 1.76 ± 1.05a 6.50 ± 2.02a 1.99 ± 0.60 e e e
Statistically significant difference at P < 0.05.
pyometra samples. Of the 28 remaining fractions, ten (with molecular weights of 125.64 kDa, 106.82 kDa, 86.31 kDa, 72.47 kDa, 46.54 kDa, 42.19 kDa, 36.31 kDa, 27.82 kDa, 25.53 kDa, and 25.01 kDa) demonstrated significantly higher (P < 0.05) intensity of staining in the pyometra tissues than in the health controls. In the case of five protein fractions (having molecular weight 147.03 kDa, 96.53 kDa, 79.54 kDa, 55.68 kDa, and 52.82 kDa) a significantly higher (P < 0.05) intensity of staining was observed in healthy uterine samples compared to pyometra samples (Fig. 1 e MAL e B, Table 3). 4. Discussion In the present study the effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs, using western blotting with selected lectins (SNA and MAL II), was investigated.
Molecular weight (kDa)
182.89 158.76 147.03 138.62 133.67 125.64 118.05 106.82 101.28 96.53 97.24 94.95 86.31 79.54 75.85 74.52 72.47 70.21 65.21 62.70 57.46 55.68 52.82 51.12 49.98 46.54 42.19 36.31 27.82 25.53 25.01 a
Densitometric analysis (arbitrary units) Pyometra group (n ¼ 10)
Control (n ¼ 10)
1.42 ± 0.92 2.17 ± 2.00 3.62 ± 1.87 2.30 ± 2.02 1.91 ± 1.56 3.28 ± 1.80 4.89 ± 2.11 8.43 ± 0.66 7.77 ± 2.53 2.75 ± 0.50 e 5.95 ± 3.39 11.90 ± 1.83 4.45 ± 1.06 4.65 ± 2.03 4.21 ± 2.01 3.90 ± 2.13 3.75 ± 1.40 3.86 ± 1.56 1.97 ± 0.84 4.29 ± 2.89 3.81 ± 2.32 2.35 ± 1.27 2.11 ± 0.86 1.26 ± 0.91 2.15 ± 1.16 5.63 ± 3.12 18.21 ± 5.10 1.21 ± 0.11 2.65 ± 0.23 1.51 ± 0.70
2.14 ± 1.10 3.13 ± 3.53 6.76 ± 2.28a 2.46 ± 1.94 2.35 ± 0.31 1.54 ± 0.32a 4.39 ± 1.39 5.63 ± 1.07a 9.16 ± 5.40 9.43 ± 7.18a 2.00 ± 1.00 4.84 ± 2.12 8.63 ± 1.97a 7.56 ± 1.66a e 6.10 ± 2.90 0.40 ± 0.19a 4.13 ± 0.40 3.18 ± 0.96 1.71 ± 0.41 5.41 ± 0.95 5.65 ± 0.89a 4.40 ± 2.20a e e 0.94 ± 0.30a 1.40 ± 0.10a 1.25 ± 0.66a 0.76 ± 0.48a 0.91 ± 1.01a 0.19 ± 0.16a
Statistically significant difference at P < 0.05.
The comparison between uterine tissues from healthy female dogs and dogs with pyometra showed different patterns of distribution of sialic acid moieties conjugated to different protein molecules. Generally, reactivity to SNA and MAL II was markedly increased in uterine tissues from female dogs with pyometra. Moreover, the intensity of staining of similar fractions markedly differed. These findings indicate that proteins in the uterine tissues from female dogs with pyometra are differently glycosylated compared to the normal uterine tissues. These changes may influence appropriate function of uterine cells and lead to variety of pathological reactions including cell communication/signaling. Lectin blotting is an extension of western blotting that uses lectin instead of antibody to detect glycoconjugates [27]. This method was used previously for the detection of altered glycosylation due to neoplastic processes [12,28]. We used two lectins reacting with sialic acid, MAL II, which interacts with a2,3-linked sialic acid, and SNA, specific for a2,6-linked sialic acid [29,30]. Sialic acids are widely distributed in nature as terminal sugars of oligosaccharides attached to proteins or lipids [31]. It has been found that sialic acids have many important functions in organism involving protection of cell membranes and molecules from attack by proteases or glycosidases, protein stabilization, binding and transport of ions and drugs, and the increase in viscosity of mucin [32,33]. Very few studies have addressed glycosylation in uterine tissues from female dogs with pyometra. In one study lectin histochemistry has been used to investigate lectin binding patterns in normal canine endometrium and endometrium from female dogs with pyometra and cystic endometrial hyperplasia [21]. The authors of the cited work found that normal endometrium showed cycledependent changes in Glycine max agglutinin (SBA), Arachis hypogaea agglutinin (PNA), Helix pomatia agglutinin (HPA) and Ulex
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europaeus agglutinin (UEA I) binding during diestrus and anoestrus. Endometrium with inflammatory alternations lost cycle-specific lectin binding patterns and, with increasing severity of pathological changes, demonstrated a marked decrease in binding intensity to the glandular and surface epithelial glycocalyx and secretion. These authors hypothesized that decreased lectin binding in uterine samples from dogs with pyometra could be consequence of bacterial occupation of lectin receptors. In turn, Bartel et al. [22] found different pattern of binding Wheat Germ agglutinin (WGA), HPA and UEA I by normal uterine endometrium and endometrial cysts. They found differences in lectin binding between surface epithelium and internal epithelium of the cysts and generally weaker binding in epithelium of endometrial cysts than in uterine endometrium. Walter et al. [19] used lectin histochemistry to investigate changes of glycoconjugates in endometrial tissues obtained from mares with chronic endometrial degeneration. They found that glycoconjugate patterns of uterine glands were altered in mares with chronic endometrial degeneration. Generally lectin (HPA, Ricinus communis agglutinin I (RCA I), UEA I and WGA) binding patterns of cystic dilated glands in endometrial samples were remarkably strong, whereas normal surrounding cells remained unstained. Many different lectins have been used in studies on tissue samples obtained from human uterus [15]. These studies have shown changes in glycosylation at different stages of the menstrual cycle, during pregnancy as well as in the course of various pathological processes in human uterus [12,15,16,20,34]. Post-translational modifications are reactions that affect amino acids present in protein chain. Depending on properties of particular amino acids different moieties can be attached and in result differently influence biological activity of protein [35]. Glycosylation is one of past-translational modifications of proteins and plays an important role in the development and physiology of living organisms but may also contribute to diseases [8]. During posttranslational glycosylation glycans can be attached to proteins either via an amide group (N-linked glycosylation) or a hydroxyl group (O-linked glycosylation). Most of the proteins in the body are N-glycosylated [35]. A number of N-linked glycoprotein changes have been identified in association with different diseases in humans including various inflammatory conditions, cancer, neurodegenerative disorders, liver diseases and diabetes mellitus [36e40]. It is known that changes in glycosylation of glycoprotein often cause a change in their function and such changes may be related with diseases [9,41]. Post-translational glycosylation can affect many cellular processes, such as cell-cell and cell-matrix recognition, adhesion, motility, control of membrane permeability and molecular recognition [41]. Same reports suggest that glycosylation may affect recruitment of neutrophils to sites of inflammation as well as binding of microorganisms during infection [42e44]. Having this data in mind it can be assumed that increased glycosylation found in the present study in the uterine tissues from dogs with pyometra was caused by some mediators produced during pyometra and may play a role in the pathogenesis and course of this disease. Because studies have shown that progesterone has an inhibitory effect on endometrial carbohydrate expression [45,46] in our study both groups of dogs were in the same phase of oestrus cycle. Thus hormonal status of studied dogs did not affect the results. In addition, in the present study protein pattern of examined tissues was also evaluated. Densitometric analysis of SDS-PAGE showed that the protein composition of the uterine tissues from dogs with pyometra differed from that of the healthy uterus. These findings confirm the results in lectin blotting obtained in our study. Some protein fractions were only detected in the uterus of female
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dogs with pyometra. Further studies are needed to identify these protein fractions. Previous studies identified variations in the uterine protein expression in different uterine diseases in women [47]. Our results are consistent with the results of studies on gene expression in the uterus from female dogs with pyometra [48e50]. These studies have found that the expression some genes differed between pyometra and healthy tissues. Our study has some limitation. We could not completely exclude any effect of age and breed of dogs on the results obtained. All the animals included in the study were selected from the group of patients of the Department of Animal Reproduction, Faculty of Veterinary Medicine in Lublin; therefore, we were unable to select the animals of the same age and breed. It should be noted, however, that some of the data from human medicine indicate that age has no effect on glycosylation of proteins [51]. In conclusion, the results of our study demonstrated increased reactivity to SNA and MAL II in uterine tissues from female dogs with pyometra compared to normal uterine tissues. These findings indicate that unidentified proteins in uterine tissues from female dogs with pyometra are differently glycosylated compared to normal uterine tissues. Our study provide the basis for further studies of the possible role of glycosylation in the pathogenesis of canine pyometra. Declaration of interest The authors declare that there is no conflict of interests regarding the publication of this article. References [1] Smith FO. Canine pyometra. Theriogenology 2006;66:610e2. [2] Jitpean S, Hagman R, Holst BS, Hoglund OV, Pettersson A, Egenvall A. Breed variations in the incidence of pyometra and mammary tumours in Swedish dogs. Reprod Domest Anim 2012;47:347e50. [3] Blendinger K, Bostedt H, Hoffmann B. Hormonal effects of the use of an antiprogestin in the bitches with pyometra. J Reprod Fertil 1997;(Suppl 5): 317e25. [4] Egenvall A, Hagman R, Bonnett BN, Hedhammar A, Olson P, Lagerstedt AS. Breed risk of pyometra in insured dogs in Sweden. J Vet Intern Med 2001;15: 530e8. [5] De Bosschere H, Ducatelle R, Vermeirsch H, Van Den Broeck W, Coryn. Cystic endometrial hyperplasia-pyometra complex in the bitch: should the two entities be disconnected? Theriogenology 2001;55:1509e19. [6] Noakes DE, Dhaliwal GK, England GC. Cystic endometrial hyperplasia/pyometra in dogs: a review of the causes and pathogenesis. J Reprod Fertil 2001;57(Suppl 200):395e406. [7] Hagman R. Canine pyometra. What is new? Reprod Domest Anim 2017;52(Suppl. 2):288e92. [8] Ohtsubo K, Marth JD. Glycosylation in cellular mechanisms of health and disease. Cell 2006;126:855e66. [9] Durand G, Seta N. Protein glycosylation and diseases: blood and urinary oligosaccharides as markers for diagnosis and therapeutic monitoring. Clin Chem 2000;46:795e805. [10] Hakomori S. Glycosylation defining cancer malignancy: new wine in an old bottle. Proc Natl Acad Sci USA 2002;99:10231e3. [11] Hirabayashi J. Concept, strategy and realization of lectin-based glycan profiling. J Biochem 2008;144:139e47. [12] Kim HJ, Kim SC, Ju W, Kim YH, Yin SY, Kim HJ. Aberrant sialylation and fucosylation of intracellular proteins in cervical tissue are critical markers of cervical carcinogenesis. Oncol Rep 2014;31:1417e22. [13] Wi GR, Moon BI, Kim HJ, Lim W, Lee A, Lee JW, Kim HJ. A lectin-based approach to de-tecting carcinogenesis in breast tissue. Oncol Lett 2016;11: 3889e95. [14] Hashim OH, Jayapalan JJ, Lee CS. Lectins: an effective tool for screening of potential cancer biomarkers. PeerJ 2017;5:e3784. https://doi.org/10.7717/ peerj.3784. [15] Clark GF. Functional glycosylation in the human and mammalian uterus. Fertil Res Pract 2015;1:17e28. [16] Lopez-Morales D, Reyes-Leyva J, Santos-Lopez G, Zenteno E, Vallejo-Ruiz V. Increased ex-pression of sialic acid in cervical biopsies with squamous intraepithelial lesions. Diagn Pathol 2010;5:1e5. [17] Sheta R, Bachvarov D. Role of aberrant glycosylation in ovarian cancer dissemination. Biomed Rev 2014;25:83e92. [18] Miller DL, Jones CJ, Aplin JD, Nardo LG. Altered glycosylation in peri-
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[36] Gornik O, Lauc G. Glycosylation of serum proteins in inflammatory diseases. Dis Markers 2008;25:267e78. [37] Pinho SS, Reis CA. Glycosylation in cancer: mechanisms and clinical implications. Nat Rev Canc 2015;15:540e55. [38] Abou-Abbass H, Abou-El-Hassan H, Bahmad H, Zibara K, Zebian A, Youssef R, Ismail J, Zhu R, Zhu S, Dong X, Nasser M, Bahmad M, Darwish H, Mechref Y, Kobeissy F. Glycosylation and other PTMs alterations in neurodegenerative diseases: current status and future role in neurotrauma. Electrophoresis 2016;37:1549e61. [39] Blomme B, Van Steenkiste C, Callewaert N, Van Vlierberghe H. Alteration of protein glyco-sylation in liver diseases. J Hepatol 2009;50:592e603. [40] Testa R, Vanhooren V, Bofigli AR, Boemi M, Olivieri F, Ceriello A, Genovese S, Spazzafu-mo L, Borelli V, Bacalini MG, Salvioli S, Garagnani P, Devaele S, Libert C, Franceschi C. N-glycomic changes in serum proteins in type 2 diabetes mellitus correlate with complications and with metabolic syndrome parameters. PLoS One 2015;10:e0119983. https://doi.org/10.1371/journal.pone. 0119983. [41] Tian Y, Zhang H. Glycoproteomics and clinical applications Proteomics. Clin Appl 2010;4:124e32. [42] Karlsson KA. Microbial recognition of target-cell glycoconjugates. Curr Opin Struct Biol 1995;5:622e35. [43] Lasky LA. Selectin-carbohydrate interactions and the initiation of inflammatory response. Annu Rev Biochem 1995;64:113e39. [44] Lowe JE. Glycan-dependent leukocyte adhesion and recruitment in inflammation. Curr Opin Cell Biol 2003;15:531e8. [45] Jones CJ, Fazleabas AT, McGinlay P, Aplin JD. Cyclic modulation of epithelial glycosylation in human and baboon (Papio Anubis) endometrium demonstrated by the binding of the agglu-tinin of Dolichos biflorus. Biol Reprod 1998;58:20e7. [46] Sen S, Chowdhury G, Chowdhury M. Sialic acid binding protein of human endometrium: its regulation by steroids. Mol Cell Biochem 2001;221:17e23. [47] Dominguez F, Santamaria J, Sanchez-Ribas I, Pellicer A. Proteomics of the endometrium and associated pathologies. mt Medecine de la Reproduction. Gynecol Endocrinol 2012;14:295e306. €nnberg E, Pejler G. Canine uterine bacterial infection induces [48] Hagman R, Ro upregulation of proteolysis-related genes and downregulation of homeobox and zinc finger factors. PLoS One 2009;4:e8039. https://doi.org/10.1371/ journal.pone.0008039. [49] Voorwald FA, Marchi FA, Villacis RAR, Alves CEF, Toniollo GH, Amorim RL, Drigo SA, Rogatto SR. Molecular expression profile reveals potential biomarkers and therapeutic targets in canine endometrial lesions. PLoS One 2015;10:e0133894. https://doi.org/10.1371/journal.pone.0133894. [50] Du M, Wang X, Yue YW, Zhou PY, Yao W, Li X, Ding XB, Liu XF, Guo H, Ma WZ. Selec-tion of reference genes in canine uterine tissues. Genet Mol Res 2016;15. https://doi.org/10.4238/gmr.15028138. [51] Kabadi UM. Glycosylation of proteins. Lack of influence of aging. Diabetes Care 1988;11:429e32.
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The effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs Marek Szczubiał a, *, Jacek Wawrzykowski b, Roman Da˛browski a, Mariola Bochniarz a, Piotr Brodzki a, Marta Kankofer b a b
Department and Clinic of Animal Reproduction, Faculty of Veterinary Medicine, University of Life Sciences, Głeboka 30, 20-612, Lublin, Poland Department of Animal Biochemistry, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12, 20-033, Lublin, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2019 Received in revised form 20 March 2019 Accepted 24 March 2019 Available online 25 March 2019
The main aim of this study was to investigate the effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs, using western blotting with selected lectins (Sambucus nigra agglutinin - SNA and Maackia amurensis agglutinin e MAL II). In addition protein pattern of examined tissues was also evaluated. The study was performed on 10 female dogs undergoing ovariohysterectomy because of pyometra and 10 clinically healthy female dogs, undergoing elective spaying (ovariohysterectomy). Uterine tissue samples of 1 cm2 were taken from the middle region of each uterine horn in both group of animals immediately after ovariohysterectomy. Tissue samples were homogenized and analysed by sodium dodecyl sulphateepolyacrylamide gel electrophoresis (SDS-PAGE) and western blotting with SNA and MAL II. SDS-PAGE analysis showed differences between pyometra samples and controls in the amount of obtained protein fractions and the protein content in the individual fractions. Five protein (with a molecular weight of 193.78 kDa, 103.18 kDa, 77.67 kDa, 70.39 kDa, and 53.00 kDa) were found only in the pyometra samples. The remaining fractions differed in intensity of staining, which indicated differ abundance of a given protein. The results of western blotting with SNA and MAL II demonstrated that the pattern obtained from densitometric analysis differs between adequate healthy and pyometra samples with regard to the amount of protein fraction obtained as well as the intensity of staining of particular fraction. The pyometra tissues contained seven SNA-binding proteins (with a molecular weight 189.94 kDa, 165.51 kDa, 100.94 kDa, 59.42 KDa, 41.32 kDa, 35.16 kDa, and 32.6 kDa) that were not in the healthy tissues. Of the nine remaining fractions, six showed significantly higher (P < 0.05) intensity of staining in the healthy uterine tissues. In turn, the MAL II-binding protein with a molecular weight 75.85 kDa, 51.12 kDa, and 49.98 kDa were found only in the pyometra samples. Of the 28 remaining fractions, ten demonstrated significantly higher (P < 0.05), and five fractions had significantly lower (P < 0.05) intensity of staining in the pyometra tissues. The results obtained indicate that proteins in uterine tissues from female dogs with pyometra are differently glycosylated compared to normal uterine tissues. These findings provide the basis for further studies of the possible role of glycosylation in the pathogenesis of canine pyometra. © 2019 Elsevier Inc. All rights reserved.
Keywords: SNA MAL II Lectin blotting Pyometra Uterus Female dogs
1. Introduction Pyometra is a common, life threatening uterine disease in intact dogs [1,2]. The disease occurs mainly in geriatric female dogs in the lutheal stage of the oestrus cycle [3,4]. The pathogenesis of pyometra has not been completely established, however, it is
* Corresponding author. E-mail address: [email protected] (M. Szczubiał). https://doi.org/10.1016/j.theriogenology.2019.03.020 0093-691X/© 2019 Elsevier Inc. All rights reserved.
commonly accepted that disease results from bacterial interaction with an endometrium that has undergone pathological changes caused by progesterone and estrogens influence [5e7]. Glycosylation is the most common post-translational modification of proteins which produces different types of glycoconjugates that are typically attached to cellular proteins and lipids [8]. Such post-translational changes can affect many cellular processes, such as cell-cell and cell-matrix recognition, adhesion, motility, control of membrane permeability, and molecular recognition [9,10].
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Glycosylation can occur spontaneously but can also be the result of pathological processes. To analyze glycosylation in normal and pathologically changed tissues many different techniques using lectins, including western blotting, can be used [11e14]. Lectins are glycan binding proteins that are typically, highly selective for specific glycan structures and for that reason are useful in the study of glycan variation [11]. It is known that the mammalian uterus is highly enriched in glycoconjugates that are associated with the apical surfaces of epithelial cells and the secretions released by both epithelium and stromal cells [15]. These glycoconjugates interact primarily with sperm, the implanting embryo, the fetus, and any pathogens that gain entry into the uterus. According to many studies, mainly from human medicine, various pathological processes related with reproduction, such as ovarian and cervical cancer, endometriosis, endometrial degeneration, different hypertensive disorders of pregnancy as well as preterm birth and unexplained infertility, are associated with alternations in glycosylation patterns [12,15e20]. Although many different lectin binding studies have been performed on tissue samples from the human uterus there are very few studies concerning uterine tissues from female dogs [21,22]. Thus, the main aim of this study was to investigate the effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs, using western blotting with selected lectins (SNA and MAL II). In addition protein pattern of examined tissues was also evaluated. 2. Material and methods 2.1. Animals The study was performed in accordance with national animal protection regulations (Animal Experimentation Act dated 15 January 2015) which are in agreement with European legislation about ethics in animal experiments. The study was performed on 20 female dogs of various breeds and mixed-breed undergoing ovariohysterectomy at the Department of Animal Reproduction, Faculty of Veterinary Medicine in Lublin. The first group involved 10 clinically healthy female dogs, aged 2e4 years, weighted 10e17 kg, between 3 and 6 weeks after oestrus, admitted to clinic for elective spaying (ovariohysterectomy) on the owners request. These dogs were classified as healthy after complete physical examination, haematology, biochemistry, and ultrasonography. The second group included 10 female dogs aged 6e12 years, weighted 12e24 kg, between 4 and 7 weeks after oestrus, undergoing ovariohysterectomy because of pyometra. These dogs were diagnosed with pyometra after medical history, physical examination, haematology, biochemistry and ultrasonography. In most cases of dogs with pyometra clinical examination revealed pyrexia, polydipsia, polyuria, anorexia, apathy, and vaginal discharge [1]. Findings on abdominal ultrasound in all affected dogs were consistent with pyometra (an enlarged uterus filled with anechoic to hypoechoic fluid with a thickened wall) [23]. Dogs with some other reproductive or other abdominal changes observed in the abdominal ultrasonography were excluded from the study. To confirm the same phase of oestrus cycle (dioestrus) in both group of dogs vaginal cytology and serum progesterone concentration were performed using commercial ELISA kit (MyBioSource, Ins., San Diego, USA). All dogs were subjected to routine ovariohysterectomy. Normal uterus and pyometra cases were confirmed by postoperative histopathological examination of the uterus by routine histopathological examination. In all cases of pyometra samples of uterine pus were collected
directly from the infected uterine horns for bacterial identification. 2.2. Tissue collection Uterine tissue samples of 1 cm2 were taken from the middle region of each uterine horn in both group of animals immediately after ovariohysterectomy. Luminal exude was excluded from endometrial samples. Tissue samples were homogenized in phosphate buffer (0.1 mol/L, pH 7.0) by use of Ultra Turrax (Ikawerk, Janke, Kunkel, Staufen, Germany), centrifuged at 4 C for 20 min at 6500 g and frozen at 76 C for further analysis. 2.3. Electrophoresis Uterine homogenates were separated by sodium dodecyl sulphateepolyacrylamide gel electrophoresis (SDS-PAGE) [24] on 16.5% polyacrylamide gels (7 5 cm, 1.5 mm), using Mini-PROTEAN Tetra cell system (Bio-Rad, Warszawa, Poland). The amount of protein loaded per each well was the same and amounted 4 mg. The running buffer was 0.025 M Tris/glycine, pH 8.3. Constant voltage of 200 V was used. After electrophoresis, control gels were stained for protein with Roti-Blue colloidal Coomassie brilliant staining (Roti, Karlsruhe, Germany). 2.4. Western blotting Protein fractions were transferred from SDS-PAGE to ImmunBlot PVDF membranes (0.2 mm, polyvinylidene difluoride; BioRad) in accordance with procedure described by Towbin et al. [25] (Criterion blotter (Bio-Rad, Warszawa, Poland), during 2 h at 400 V in 4 C). To eliminate non-specific immune response, membranes were incubated for 1 h in 15 ml 0.05% Tween 20 in R.T.U. animal free block solution (SP-5035, Vector Laboratories, Burlingame, CA, USA). Biotinylated lectins were used as antibodies for appropriate sugar moieties in uterine proteins. Overnight incubation at 4 C with biotinylated Maackia Amurensis lectin II, MAL II (B1265, Vector Laboratories) for glycans that bind sialic acid in a 2,3linkage (1 mg/mL in 0,05% PBST) was performed and respectively overnight incubation 4 C with biotinylated Sambucus Nigra lectin, SNA (B-1305, Vector Laboratories) for glycans that bind sialic acid in a 2,6-linkage (1 mg/mL in 0,05% PBST). Complexes of biotinylated lectins with appropriate glycoproteins were visualized by staining with streptavidin-based conjugated with alkaline phosphatase with the use of reagent kit (VECTASTAIN ABC-AP Staining KIT, AK-5000 and BCIP/NBT Alkaline Phosphatase (AP) Substrate Kit, SK-5400, Vector Laboratories) in accordance with the manufacturer’s procedure. Dark fractions located at selected molecular weight positions were considered as positive reactions. As a positive and negative control, horseradish peroxidase (Peroxidase from horseradish, 77332, Sigma) and trypsin inhibitor (Trypsin inhibitor from Glycine max (soybean), T9003, Sigma) were used on each gel respectively. The membranes were scanned on an imaging densitometer GS710 (Bio-Rad, Warszawa, Poland), and the molecular weight and relative quantities were estimated with the Bio-Rad Quantity One 4.1 software (Bio-Rad). Protein content in the samples was determined according to the method based on the biuret reaction using a commercial colorimetric kit (Cormay, Lublin, Poland) [26]. 2.5. Statistical analysis The results were presented as means ± SD and analysed using the computer program STATISTICA version 10.0 (Statsoft, USA).
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After testing for normal distribution (the Shapiro-Wilk/ Kolmogorov-Smirnov tests), the Student’s t-test was used to compare the results of densitometric analysis between the pyometra and control groups. Differences were considered significant at P < 0.05. 3. Results Microbiological examination identified Escherichia coli in the samples of uterine pus from all female dogs with pyometra. To compare binding patterns of SNA and MAL II in uterus from healthy female dogs and uterus from female dogs with pyometra, 10 individual homogenates of normal uterine tissues and 10 homogenates of diseased uterine tissues were examined by western blotting using these two lectins. Fig. 1 shows the banding patterns of proteins after sodium dodecyl sulphate epolyacrylamide gel electrophoresis (SDS-PAGE) and after western blotting with SNA and MAL II in tissue homogenates from uterus of healthy female dogs and uterus from dogs with pyometra. Densitometric analysis of SDS-PAGE gels showed that the protein composition of the uterine tissues from dogs with pyometra differed from that of the healthy uterus. This analysis demonstrated differences in the amount of protein fractions obtained and the protein content in the individual fractions as expressed by alterations in staining intensity of fraction. The protein with a molecular weight of 193.78 kDa as well as the proteins with molecular weights of 103.18 kDa, 77.67 kDa, 70.39 kDa, and 53.00 kDa were found only in the uterine samples from dogs with pyometra (Fig. 1 e SDS-PAGE separation, Table 1). The remaining fractions differed in intensity of staining, which indicated different abundance of a given protein. In case of proteins with molecular weights of 117.77 kDa, 98.19 kDa, 89.16 kDa, 75.36 kDa, 67.07 kDa, 42.23 kDa, 35.37 kDa, 33.35 kDa, 29.97 kDa and 25.59 kDa differences in intensity of staining reached statistical significance (P < 0.05) (Fig. 1 e SDS-PAGE separation, Table 1). Results of western blotting with SNA and MAL II clearly demonstrated that the pattern obtained from densitometric analysis differed between adequate healthy and pyometra samples with regard to the number of protein fractions obtained as well as the intensity of staining of particular fraction. Generally, the homogenates from dogs with pyometra had markedly increased reactivity to SNA. The pyometra tissues contained seven SNA-binding proteins (with molecular weights of 189.94 kDa, 165.51 kDa, 100.94 kDa, 59.42 KDa, 41.32 kDa, 35.16 kDa, and 32.6 kDa) that were not in the healthy tissues (Fig. 1 e SNA e A, Table 2). Of the nine remaining fractions, five (with molecular weights of 147.92 kDa, 92.14 kDa, 85.20 kDa, 74.91 kDa, and 54.53 kDa) showed significantly higher (P < 0.05) intensity of staining in healthy uterine tissues than in compared pyometra cases. The fraction with a molecular weight 49.77 kDa intensity of staining was significantly higher (P < 0.05) in pyometra samples (Fig. 1 e SNA e B, Table 2). Also proteins in the pyometra samples tended to bind more MAL II and a higher intensity of band staining was observed in these samples. The MAL II-binding proteins with molecular weights of 75.85 kDa, 51.12 kDa, and 49.98 kDa were found only in the
Fig. 1. The patterns of electrophoretic and western blotting separations from uterine samples of pyometra and healthy bitches. Upper pattern e electrophoretic line and its densitometric analysis from SDS-PAGE separation; middle pattern e western blotting pattern and its densitometric analysis from SNA determinations; bottom pattern western blotting pattern and its densitometric analysis from MALL II determinations. (A) e pyometra samples, (B) e control samples. Bottom part of each picture represents example of most representative paths while upper part shows mean results of densitometric analysis of all adequate paths and fractions.
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Table 1 The results of densitometric analysis of SDS-PAGE gels. Molecular weight (kDa)
193.78 165.64 137.31 128.25 117.77 106.02 103.18 98.19 94.20 89.16 77.67 75.36 70.91 70.39 67.07 58.52 53.00 49.75 42.23 39.23 37.13 35.37 33.35 29.97 25.59 a
Table 3 The results of densitometric analysis of MALL II blottings.
Densitometric analysis (arbitrary units) Pyometra group (n ¼ 10)
Control (n ¼ 10)
1.09 ± 0.37 2.14 ± 1.02 0.93 ± 0.47 1.26 ± 0.41 0.95 ± 0.27 1.73 ± 0.72 2.82 ± 0.29 1.27 ± 0.29 1.28 ± 0.70 4.41 ± 1.52 5.10 ± 3.10 0.80 ± 0.33 15.15 ± 9.96 0.92 ± 0.25 19.21 ± 2.93 2.59 ± 0.95 1.41 ± 0.41 7.16 ± 2.06 11.04 ± 1.81 3.23 ± 0.73 2.21 ± 0.81 3.97 ± 1.11 6.51 ± 1.55 2.45 ± 0.72 8.29 ± 6.98
e 1.54 ± 0.36 0.78 ± 0.22 0.98 ± 0.62 0.57 ± 0.15a 1.67 ± 0.37 e 0.48 ± 0.06a 1.60 ± 0.40 1.11 ± 0.08a e 6.50 ± 1.02a e e 28.29 ± 3.52a 2.20 ± 0.41 e 6.06 ± 1.83 14.56 ± 1.10a 3.28 ± 0.37 2.68 ± 0.73 2.58 ± 0.90a 3.57 ± 0.86a 1.02 ± 0.33a 4.26 ± 2.06a
Statistically significant difference at P < 0.05.
Table 2 The results of densitometric analysis of SNA blottings. Molecular weight (kDa)
189.94 165.51 147.92 117.19 100.94 92.14 85.20 74.91 65.29 59.42 54.53 49.77 43.26 41.32 35.16 32.60 a
Densitometric analysis (arbitrary units) Pyometra group (n ¼ 10)
Control (n ¼ 10)
13.4 ± 9.84 6.40 ± 2.64 9.03 ± 2.71 23.36 ± 3.85 19.15 ± 6.27 2.03 ± 1.85 6.98 ± 3.63 2.08 ± 0.27 11.85 ± 6.31 1.66 ± 1.26 0.55 ± 0.07 4.07 ± 1.39 2.37 ± 1.53 4.65 ± 1.98 2.55 ± 0.27 1.66 ± 1.51
e e 20.11 ± 6.93a 19.55 ± 8.71 e 16.77 ± 2.48a 13.98 ± 5.97a 4.57 ± 0.81a 8.31 ± 1.33 1.76 ± 1.05a 6.50 ± 2.02a 1.99 ± 0.60 e e e
Statistically significant difference at P < 0.05.
pyometra samples. Of the 28 remaining fractions, ten (with molecular weights of 125.64 kDa, 106.82 kDa, 86.31 kDa, 72.47 kDa, 46.54 kDa, 42.19 kDa, 36.31 kDa, 27.82 kDa, 25.53 kDa, and 25.01 kDa) demonstrated significantly higher (P < 0.05) intensity of staining in the pyometra tissues than in the health controls. In the case of five protein fractions (having molecular weight 147.03 kDa, 96.53 kDa, 79.54 kDa, 55.68 kDa, and 52.82 kDa) a significantly higher (P < 0.05) intensity of staining was observed in healthy uterine samples compared to pyometra samples (Fig. 1 e MAL e B, Table 3). 4. Discussion In the present study the effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs, using western blotting with selected lectins (SNA and MAL II), was investigated.
Molecular weight (kDa)
182.89 158.76 147.03 138.62 133.67 125.64 118.05 106.82 101.28 96.53 97.24 94.95 86.31 79.54 75.85 74.52 72.47 70.21 65.21 62.70 57.46 55.68 52.82 51.12 49.98 46.54 42.19 36.31 27.82 25.53 25.01 a
Densitometric analysis (arbitrary units) Pyometra group (n ¼ 10)
Control (n ¼ 10)
1.42 ± 0.92 2.17 ± 2.00 3.62 ± 1.87 2.30 ± 2.02 1.91 ± 1.56 3.28 ± 1.80 4.89 ± 2.11 8.43 ± 0.66 7.77 ± 2.53 2.75 ± 0.50 e 5.95 ± 3.39 11.90 ± 1.83 4.45 ± 1.06 4.65 ± 2.03 4.21 ± 2.01 3.90 ± 2.13 3.75 ± 1.40 3.86 ± 1.56 1.97 ± 0.84 4.29 ± 2.89 3.81 ± 2.32 2.35 ± 1.27 2.11 ± 0.86 1.26 ± 0.91 2.15 ± 1.16 5.63 ± 3.12 18.21 ± 5.10 1.21 ± 0.11 2.65 ± 0.23 1.51 ± 0.70
2.14 ± 1.10 3.13 ± 3.53 6.76 ± 2.28a 2.46 ± 1.94 2.35 ± 0.31 1.54 ± 0.32a 4.39 ± 1.39 5.63 ± 1.07a 9.16 ± 5.40 9.43 ± 7.18a 2.00 ± 1.00 4.84 ± 2.12 8.63 ± 1.97a 7.56 ± 1.66a e 6.10 ± 2.90 0.40 ± 0.19a 4.13 ± 0.40 3.18 ± 0.96 1.71 ± 0.41 5.41 ± 0.95 5.65 ± 0.89a 4.40 ± 2.20a e e 0.94 ± 0.30a 1.40 ± 0.10a 1.25 ± 0.66a 0.76 ± 0.48a 0.91 ± 1.01a 0.19 ± 0.16a
Statistically significant difference at P < 0.05.
The comparison between uterine tissues from healthy female dogs and dogs with pyometra showed different patterns of distribution of sialic acid moieties conjugated to different protein molecules. Generally, reactivity to SNA and MAL II was markedly increased in uterine tissues from female dogs with pyometra. Moreover, the intensity of staining of similar fractions markedly differed. These findings indicate that proteins in the uterine tissues from female dogs with pyometra are differently glycosylated compared to the normal uterine tissues. These changes may influence appropriate function of uterine cells and lead to variety of pathological reactions including cell communication/signaling. Lectin blotting is an extension of western blotting that uses lectin instead of antibody to detect glycoconjugates [27]. This method was used previously for the detection of altered glycosylation due to neoplastic processes [12,28]. We used two lectins reacting with sialic acid, MAL II, which interacts with a2,3-linked sialic acid, and SNA, specific for a2,6-linked sialic acid [29,30]. Sialic acids are widely distributed in nature as terminal sugars of oligosaccharides attached to proteins or lipids [31]. It has been found that sialic acids have many important functions in organism involving protection of cell membranes and molecules from attack by proteases or glycosidases, protein stabilization, binding and transport of ions and drugs, and the increase in viscosity of mucin [32,33]. Very few studies have addressed glycosylation in uterine tissues from female dogs with pyometra. In one study lectin histochemistry has been used to investigate lectin binding patterns in normal canine endometrium and endometrium from female dogs with pyometra and cystic endometrial hyperplasia [21]. The authors of the cited work found that normal endometrium showed cycledependent changes in Glycine max agglutinin (SBA), Arachis hypogaea agglutinin (PNA), Helix pomatia agglutinin (HPA) and Ulex
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europaeus agglutinin (UEA I) binding during diestrus and anoestrus. Endometrium with inflammatory alternations lost cycle-specific lectin binding patterns and, with increasing severity of pathological changes, demonstrated a marked decrease in binding intensity to the glandular and surface epithelial glycocalyx and secretion. These authors hypothesized that decreased lectin binding in uterine samples from dogs with pyometra could be consequence of bacterial occupation of lectin receptors. In turn, Bartel et al. [22] found different pattern of binding Wheat Germ agglutinin (WGA), HPA and UEA I by normal uterine endometrium and endometrial cysts. They found differences in lectin binding between surface epithelium and internal epithelium of the cysts and generally weaker binding in epithelium of endometrial cysts than in uterine endometrium. Walter et al. [19] used lectin histochemistry to investigate changes of glycoconjugates in endometrial tissues obtained from mares with chronic endometrial degeneration. They found that glycoconjugate patterns of uterine glands were altered in mares with chronic endometrial degeneration. Generally lectin (HPA, Ricinus communis agglutinin I (RCA I), UEA I and WGA) binding patterns of cystic dilated glands in endometrial samples were remarkably strong, whereas normal surrounding cells remained unstained. Many different lectins have been used in studies on tissue samples obtained from human uterus [15]. These studies have shown changes in glycosylation at different stages of the menstrual cycle, during pregnancy as well as in the course of various pathological processes in human uterus [12,15,16,20,34]. Post-translational modifications are reactions that affect amino acids present in protein chain. Depending on properties of particular amino acids different moieties can be attached and in result differently influence biological activity of protein [35]. Glycosylation is one of past-translational modifications of proteins and plays an important role in the development and physiology of living organisms but may also contribute to diseases [8]. During posttranslational glycosylation glycans can be attached to proteins either via an amide group (N-linked glycosylation) or a hydroxyl group (O-linked glycosylation). Most of the proteins in the body are N-glycosylated [35]. A number of N-linked glycoprotein changes have been identified in association with different diseases in humans including various inflammatory conditions, cancer, neurodegenerative disorders, liver diseases and diabetes mellitus [36e40]. It is known that changes in glycosylation of glycoprotein often cause a change in their function and such changes may be related with diseases [9,41]. Post-translational glycosylation can affect many cellular processes, such as cell-cell and cell-matrix recognition, adhesion, motility, control of membrane permeability and molecular recognition [41]. Same reports suggest that glycosylation may affect recruitment of neutrophils to sites of inflammation as well as binding of microorganisms during infection [42e44]. Having this data in mind it can be assumed that increased glycosylation found in the present study in the uterine tissues from dogs with pyometra was caused by some mediators produced during pyometra and may play a role in the pathogenesis and course of this disease. Because studies have shown that progesterone has an inhibitory effect on endometrial carbohydrate expression [45,46] in our study both groups of dogs were in the same phase of oestrus cycle. Thus hormonal status of studied dogs did not affect the results. In addition, in the present study protein pattern of examined tissues was also evaluated. Densitometric analysis of SDS-PAGE showed that the protein composition of the uterine tissues from dogs with pyometra differed from that of the healthy uterus. These findings confirm the results in lectin blotting obtained in our study. Some protein fractions were only detected in the uterus of female
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dogs with pyometra. Further studies are needed to identify these protein fractions. Previous studies identified variations in the uterine protein expression in different uterine diseases in women [47]. Our results are consistent with the results of studies on gene expression in the uterus from female dogs with pyometra [48e50]. These studies have found that the expression some genes differed between pyometra and healthy tissues. Our study has some limitation. We could not completely exclude any effect of age and breed of dogs on the results obtained. All the animals included in the study were selected from the group of patients of the Department of Animal Reproduction, Faculty of Veterinary Medicine in Lublin; therefore, we were unable to select the animals of the same age and breed. It should be noted, however, that some of the data from human medicine indicate that age has no effect on glycosylation of proteins [51]. In conclusion, the results of our study demonstrated increased reactivity to SNA and MAL II in uterine tissues from female dogs with pyometra compared to normal uterine tissues. These findings indicate that unidentified proteins in uterine tissues from female dogs with pyometra are differently glycosylated compared to normal uterine tissues. Our study provide the basis for further studies of the possible role of glycosylation in the pathogenesis of canine pyometra. Declaration of interest The authors declare that there is no conflict of interests regarding the publication of this article. References [1] Smith FO. Canine pyometra. Theriogenology 2006;66:610e2. [2] Jitpean S, Hagman R, Holst BS, Hoglund OV, Pettersson A, Egenvall A. Breed variations in the incidence of pyometra and mammary tumours in Swedish dogs. Reprod Domest Anim 2012;47:347e50. [3] Blendinger K, Bostedt H, Hoffmann B. Hormonal effects of the use of an antiprogestin in the bitches with pyometra. J Reprod Fertil 1997;(Suppl 5): 317e25. [4] Egenvall A, Hagman R, Bonnett BN, Hedhammar A, Olson P, Lagerstedt AS. Breed risk of pyometra in insured dogs in Sweden. J Vet Intern Med 2001;15: 530e8. [5] De Bosschere H, Ducatelle R, Vermeirsch H, Van Den Broeck W, Coryn. Cystic endometrial hyperplasia-pyometra complex in the bitch: should the two entities be disconnected? Theriogenology 2001;55:1509e19. [6] Noakes DE, Dhaliwal GK, England GC. Cystic endometrial hyperplasia/pyometra in dogs: a review of the causes and pathogenesis. J Reprod Fertil 2001;57(Suppl 200):395e406. [7] Hagman R. Canine pyometra. What is new? Reprod Domest Anim 2017;52(Suppl. 2):288e92. [8] Ohtsubo K, Marth JD. Glycosylation in cellular mechanisms of health and disease. Cell 2006;126:855e66. [9] Durand G, Seta N. Protein glycosylation and diseases: blood and urinary oligosaccharides as markers for diagnosis and therapeutic monitoring. Clin Chem 2000;46:795e805. [10] Hakomori S. Glycosylation defining cancer malignancy: new wine in an old bottle. Proc Natl Acad Sci USA 2002;99:10231e3. [11] Hirabayashi J. Concept, strategy and realization of lectin-based glycan profiling. J Biochem 2008;144:139e47. [12] Kim HJ, Kim SC, Ju W, Kim YH, Yin SY, Kim HJ. Aberrant sialylation and fucosylation of intracellular proteins in cervical tissue are critical markers of cervical carcinogenesis. Oncol Rep 2014;31:1417e22. [13] Wi GR, Moon BI, Kim HJ, Lim W, Lee A, Lee JW, Kim HJ. A lectin-based approach to de-tecting carcinogenesis in breast tissue. Oncol Lett 2016;11: 3889e95. [14] Hashim OH, Jayapalan JJ, Lee CS. Lectins: an effective tool for screening of potential cancer biomarkers. PeerJ 2017;5:e3784. https://doi.org/10.7717/ peerj.3784. [15] Clark GF. Functional glycosylation in the human and mammalian uterus. Fertil Res Pract 2015;1:17e28. [16] Lopez-Morales D, Reyes-Leyva J, Santos-Lopez G, Zenteno E, Vallejo-Ruiz V. Increased ex-pression of sialic acid in cervical biopsies with squamous intraepithelial lesions. Diagn Pathol 2010;5:1e5. [17] Sheta R, Bachvarov D. Role of aberrant glycosylation in ovarian cancer dissemination. Biomed Rev 2014;25:83e92. [18] Miller DL, Jones CJ, Aplin JD, Nardo LG. Altered glycosylation in peri-
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