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ISSN : 2671-4639(Print)
ISSN : 2671-4663(Online)
Journal of Animal Reproduciton and Biotechnology Vol.27 No.2 pp.93-100

Detection of Matrix Metalloprotease-9 and Analysis of Protein Patterns in Bovine Vaginal Mucus during Estrus and Pregnancy

Jong Taek Yoon1,*, Sang Hwan Kim2, Jun Seok Baek3, Ho Jun Lee2, Kwan Sik Min3, Deuk Hwan Lee1
1Department of Animal Life Science, Hankyong National University
1Department of Animal Life Science, Hankyong National University, 3Graduate School of Bio & Information Technology, Hankyong National University
received: 2012. 5. 12, revised: 2012. 5. 13, accepted: 2012. 5. 30


To investigate the biochemical nature of changes in vaginal physiology during estrus and pregnancy, we examinedthe cytology and viscosity, and monitored the protein expression profile in vaginal mucus during estrus and pregnancy.The viscosity progressively decreased from estrus to pregnancy. Cell type analysis revealed that white blood cellsprogressively increased from estrus to pregnancy, while red blood cells progressively decreased during pregnancy. Thecornification index (CI) was higher in estrus than in pregnancy. Protein mass spectrumetry identified the presence ofribosome-binding protein 1, GRIP 1 (Glutamate receptor-interacting protein 1)-associated protein 1, DUF729 (Domainof unknown function729) domain-containing protein 1, prolactin precursor, dihydrofolatereductase, and MMP (Matrixmetalloprotease)-9 in vaginal mucus. MMP-2 and MMP-9 proteins in the vaginal mucus were active throughout estrusand gestation, as measured by a gelatinase assay, but most abundant in the vaginal mucus on day 0 of estrus. Resultsfrom ELISA of MMP-2 and MMP-9 were in accordance with the gelatinase assay. In light of the crucial role ofmetalloproteinases in extracellular matrix remodeling, the level of MMP-9 in vaginal mucus might be useful as anindicator of estrus and pregnancy to increase the efficiency of reproduction.

04 Sang Hwan Kim.pdf2.43MB


 Tissue remodeling is known to play an essential role in the estrus cycle and pregnancy. Normal progression of pregnancy depends upon the cyclical activity of hormones and pregnancy genes, and the precise timing of their expression during pregnancy is crucial for successful reproduction. Vaginal and cervical mucus plays an important role in the reproductive process in all mammals. Vaginal fluid is a biological product of complex composition that is mainly derived from cervical secretions. Physiological and biochemical properties of bovine vaginal mucus have been determined, but there is a paucity of reports about the function of these factors (Prasad et al., 1981). Using crystallization of the vaginal mucus, many workers have related ferning patterns to the stage of the estrous cycle, noting that maximal arburization occurred at the onset of estrus (Bone, 1954; Alliston et al., 1958; Fallon and Crofts, 1959; Abusineina, 1962; Noonan et al., 1975; Grobbelaar and Kay, 1985).

 Individual mucus protein components have been extensively studied to examine the physiology of pregnancy and implantation. For instance, MMP (Matrix metalloproteinase)-9 is routinely assayed for the detection of pregnancy and estrus. MMP-9 is believed to be involved in changes in the ECM (Extracellular matrix), follicle growth, and implantation (Smith et al., 1999; Woessner, 2002). MMPs, including MMP 2 and MMP-9, play an important role in tissue remodeling in various physiological processes, such as implantation and ovarian and uterine functions during estrus and pregnancy (Takagi et al., 2007). Components of the ECM can be altered through cleavage by MMPs, whose activities are inhibited by TIMPs (Tissue inhibitors of metalloproteinases) (Nagase and Woessner, 1999). While the roles of MMPs during peripartum, termination of gestation, and postpartum in cows have been reported (Walter and Boos, 2001), their expression profile during implantation has not been elucidated in detail. Thus, proteolysis mechanisms in the bovine endometrial ECM during implantation remain obscure. Most instances of bovine infertility occur during the early implantation period. The main cause of this failure usually relates to uterine circumstances, which in turn depend on humoralregulation, and perhaps on the spatiotemporal condition of the endometrium as well (Salamonsen, 1999; Curry and Osteen, 2001). Abnormalities in endometrial function cause early in fertility in many species. MMPs participate in the regulation of endometrial functions in ruminants (Riley et al., 2000), affecting the pattern of vaginal mucus proteins during estrus and pregnancy. The objective of this study was to examine the presence of MMP-9 protein and the changes in the pattern of vaginal mucus proteins during estrus and pregancy.


1. Animal Maintenance and Collection of Vaginal Mucus Samples

 Bovine vaginal mucus was collected according to the method of Chung et al. (2008). Holstein cattle (n = 23) aged 2~4 years were used. All animals had a body condition score of 4 or 5 on a1~5 scale (Rae et al., 1993). Uterine health was confirmed by cytological analysis of vaginal mucus smears. A controlled internal drug release device (CIDR) containing 1.38 g progesterone (Pfizer Animal Health, New York, NY) was inserted into 13 animals (CIDR insertion = day 0). On day 14, the device was removed and gonadotropin-releasing hormone (GnRH, Cystorelin; 100 μg; Merial, Athens, GA) was injected intramuscularly on day 15. Vaginal mucus samples were collected from animals on days 7, 30, and 210 of pregnancy, and on days 0 and 7 of estrus. 

2. Viscosity and Crystallization Patterns in Vaginal Mucus during Estrus and Pregnancy

 A sample of the collected fluid was placed onto a tilted glass slide and allowed to spread and dry at room temperature. The dried vaginal mucus was stained with Giemsa R66 (BDH Laboratory Supplies, Poole, UK).

 The stained sample was then glued onto specimen holders and examined for arborization using a scanning electron microscope (SEM)with 44.7-mA emission current for 3 min on the carbon film coater (TM-1000, Hankyong University, Ansung, Korea), at 15 kV and 5,000× magnification. Arborization analysis revealed several ferning patterns ranging from minor ferning (long stems and clear venation and subvenation) to bold ferning (short stems and irregular scattered stellate patterns) (Papanicolaou, 1946; Garm and Skjerven, 1952).

3. Analysis of Vaginal Cytology and Viscosity

 The smear was stained with GiemsaR66 and the vaginal cytology was examined. Cytological analyses of vaginal mucus samples from pregnant animals, and animals in estrus, were performed by counting white and red blood cells. The percentage of cornified epithelial cells was calculated as the “cornification index” (CI) (England and Allen, 1989). The CI was calculated by converting the total number of cornified epithelial cells in to a cellular percentage.

4. Protein Extraction and Electrophoresis

 Vaginal mucus samples were washed in wash buffer (1×PBS with 0.25% Triton X-100) and concentrated by centrifugation at 3,000 rpm for 30 s at room temperature. About 4 ml of vaginal mucus was collected (during estrus and on days 7, 30, and 210 of gestation), placed in Tris-buffered saline (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl2 , 0.05% Brij 35, 10 μg/ml leupeptin, and 1 mM PMSF), and homogenized using an Ultra-Turrax homogenizer (IKA Works, Guangzhou, China), followed by a 30 min incubation on ice to induce lysis. Finally, the tube was centrifuged at 13,000 rpm (4℃) for 5 min and the supernatant was transferred to a fresh 1.5 ml tube. The protein concentration was then measured by the Bradford method and the proteins were electrophoreticcally separated on a 12% acryl amide gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 (Bio-Rad, USA) to visualize proteins.

5. Protein Identification

 Protein bands were excised from the gel and subjected to identification by peptide mapping (Chung et al., 2008). This identification method used trypsinenzyme to digest the protein at specific sites and generate distinct peptides. Protein digestion was carried out in a clean 1.5 ml tube. The Coomassie Brilliant Blue stain was removed from excised gelpieces by washing twice with 0.5 ml of 10 mM ammonium bicarbonate in 50% acetonitrile (ACN) for 30 min. A small volume (2 to 10 ml) of 10 mM ammonium bicarbonate containing 5 pmol trypsin was added to the reconstructed gel, together with a sufficient volume of 10 mM ammonium bicarbonate buffer. Additional buffer was continuously added over a period of 2 h until the gel was saturated and just covered by buffer. After a 16 h incubation at 30℃ to allow tryptic digestion, the sample was transferred to a new tube and allowed to dry. Once the reaction buffer was completely evaporated, 2 μl of 0.1% trifluoroacetic acid in 50% acetonitrile solution was added. Mass spectrometry of the peptide solution was acquired using an Applied Biosystems 4700 Proteomics analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Norwalk, CT). The mass of the peptide was determined by the Mascot program using the SWISS-Prot and NCBI databases to search for proteins with the same score, and molecular weight and pI values were compared for confirmation.

6. Measurement of MMP-2 and MMP-9 Activity by Gelatinase Zymography

 The enzymatic activity ofMMP-2 and MMP-9 was detected by gelatinase zymography as described previously (Sato et al., 1999; Imai et al., 2003). Briefly, 10 μg protein in 10 μl Tris buffer was loaded into individual wells of a 12.5% SDS gel containing 1 mg/ml gelatin A and B, and proteins were electrophoretically separated using a mini-gel apparatus (Bio-Rad, Hercules, CA, USA) under non-reducing conditions. Following electrophoresis, gels were incubated in 2.5% (v/v) Triton X-100 for 1 h to remove SDS. Gels were rinsed 3 times (20 min per rinse) in water and incubated for 15~18 h at 37~38℃ in incubation buffer (50 mM Tris-HClpH 7.5, 10 mM CaCl2 , 5 mM ZnCl2 ) with or without 12 mM 1,10-phenanthroline (an inhibitor of MMP activity). Gels were subsequently stained with 0.5% (w/v) Coomassie Brilliant Blue R250. Proteolytic activity was observed as clear bands on a blue background. The relative molecular mass (Mr) of gelatinolytic MMP was determined by comparison with molecular weight markers (Bio-Rad) in the adjacent lane. The band intensity, representing MMP activity, was quantified by densitometry using a GS 700 Imaging Densitometer (Bio-Rad).

7. Enzyme Linked Immunosorbent Assay (ELISA) of MMP-9

 For the ELISA assay, vaginal mucus protein samples were diluted in 100% assay buffer. Vaginal mucus MMP-9 levels were measured using a quantitative sandwich ELISA test (R&D Systems, Abingdon, UK) according to the manufacturer's guidelines. All samples were measured in duplicates, and the mean was calculated. Levels of MMP-9 were determined according to a standard curve, which takes in to account 4 parameters based on the following equation: y = (A-D)/(1+(x/C)^B)+D). The standard curve was calculated from 7 known values. All MMP-9 values were reported in ng/ml.

8. Statistical Analysis

 Data were subjected to a T-test and GLM of the Statistical Analysis System (SAS Institute, version 9.4, Cary, NC, USA). Differences among treatment means were determined by using Duncan’s multiple range tests. The statistical significance was established at p<0.05.


1. Crystallization and Viscosity of Vaginal Mucus during Estrus and Pregnancy

 The crystallization of vaginal mucus during estrus was different from that seen during gestation. The bold ferning pattern became progressively denser during the progression from estrus into pregnancy (Fig. 1). The viscosity of vaginal mucus progressively increased from day 0 of estrus to day 210 of pregnancy (Fig. 2A).

Fig. 1. Scanning electron micrographs (SEM) of bovine vaginal mucus samples showing crystallization arborization. Note the dense, compact matrix arranged as crystals with rough, regular ferning. (a, c, e) Scale bar, 100 μm; (b, d, f) Scale bar, 20 μm.

Fig. 2. Analysis of Giemsa R66 staining in vaginal mucus during the estrous and pregnancy of bovine. A : Viscosity and physiological properties of vaginal mucus during estrus and pregnancy. B : Analysis of blood cells and cornification index from the vaginal mucus.

2. Cytology of Vaginal Mucus during Estrus and Pregnancy

 We observed significant cytological changes in vaginal mucus from estrus through pregnancy (Fig. 2B). The number of white blood cells progressively increased from 0 on day 0 of estrus, to 1.35 ± 1.04, and 1.60 ± 0.08, on days 30 and 210 of pregnancy, respectively. On the other hand, the number of red blood cells progressively decreased from 1.37 ± 0.28 (day 0 of estrus), to 1.15 ± 0.12 (day 30 of pregnancy) and 0.40 ± 0.40 (day 210 of pregnancy). The CI of vaginal mucus was higher during estrus (93.02 ± 27.58%), than on day 30 (49.50 ± 3.32%) and day 210 (19.90 ± 24.25%) of pregnancy (Table 1).

Table 1. Percentages of red and white blood cells, and the cornification index, in vaginal smears during estrus and pregnancy

3. Identification and Expression Profiles of Proteins in Vaginal Mucus

 The pattern of protein expression in the vaginal mucus was analyzed by SDS-PAGE electrophoresis. The levels of 16 detected proteins progressively increased from day 0 of estrus (41.76 ± 3.5 ng/ml) to day 30 (71.66 ± 2.8 ng/ml) and day 210 (102.88 ± 4.2 ng/ml) of pregnancy (Fig. 3B). A representative view of protein peptide values from the upper fraction of lanes 1~2 is shown in Fig. 3. Mass spectrophotometric analysis of 16 detected proteins revealed the identity of 6 of these proteins. The identified proteins were ribosome-binding protein 1, GRIP 1 (Glutamate receptor-interacting protein 1)-associated protein 1, DUF729 (Domain of unknown function 729) domaincontaining protein 1, prolactin precursor, dihydrofolatereductase, and MMP (Matrix metalloprotease)-9 (Table 2). As shown in Fig. 3A and 3C, 10 of the detected proteins showed an increase in expression levels as the estrus stage progressed into pregnancy. Interestingly, the expression patterns of about 40% of the detected proteins were distinct examined.

Fig. 3. SDS-PAGE profiles and MALDI-TOF mass spectrum analysis of vaginal mucus samples collected at estrus and during pregnancy.A : Protein expression patterns in vaginal mucus in estrus day 0 and pregnancy day 210. B : Concentration of extracted proteins on estrus day 0 and pregnancy days 30 and 210. Six proteins were selected for mass analysis using 4700 Proteomics Analyzer (Applied Biosystems). 1: Estrus day 0, 2: Pregnancy day 210. The asterisks indicate proteins that were not analyzed using mass spectrometry. C : MALDI-TOF mass spectrum of bovine vaginal mucus during pregnancy.

Table 2. Characterization of bovine vaginal mucus proteins differentially expressed between spontaneous and induced estrus

4. Expression of MMP-9 Protein and Gelatinolytic Activity in Vaginal Mucus during Estrus and Pregnancy

 MMP-2 and MMP-9 proteins in vaginal mucus were both activated during estrus and gestation. The activation patterns of these 2 metalloproteinases were similar during this period (Fig. 4). The active forms of MMP-2 and MMP-9 were most abundant in vaginal mucus on day 0 of estrous and lowest on day 7 of estrus. The level of the active form progressively increased on days 7, 30, and 210 in pregnancy. The gelatinase activity at Mr 72kDa and 92kDa in the vaginal mucus zymography corresponded to the latent forms of gelatinase A (MMP-2) and gelatinase B (MMP-9) (Nagase and Woessner, 1999), respectively. ELISA analysis of vaginal mucus indicated that the MMP-2 protein concentration was 2,450 ng/ml and 1,932 ± 50 ng/ml on days 0 and 7 of estrus, and 1,609 ± 35 ng/ml, 1,402 ± 32 ng/ml and 1,644 ± 19 ng/ml on days 7, 30, and 210 of pregnancy (Fig. 5A). The levels of MMP-9 in vaginal mucus were 2,390 ± 52 ng/ml and 1,856 ± 45 ng/ml on days 0 and 7 of estrus, and 1,962 ± 32 ng/ml, 1,503 ± 30 ng/ml, and 1,743 ± 31 ng/ml on days 7, 30, and 210 of pregnancy. MMP-9 production decreased as early gestation progressed, but increased again from day 30 to day 210 of gestation (Fig. 5B).

Fig. 4. Gelatinase activity in bovine vaginal mucus during estrus and pregnancy. A represent active zymography gel shows the highest activity of the MMPs present in the vaginal mucus on Day 0 of the estrous cycle and Day 210 of pregnancy. The latent form of MMP-2 is about 72 kDa and that of MMP-9 is about 92 kDa.

Fig. 5. Detection of MMP-2 and MMP-9 proteins by ELISA in bovine vaginal mucus during estrus and pregnancy. A: MMP-2, B: MMP-9. a~d Different letters within the same column represent a significant difference (p<0.05).


 During the progression from estrus to gestation, vaginal mucus undergoes changes in cytology and physiology, accompanied by characteristic crystallization and arborization patterns (England and Allen, 1989; O and Choi, 1973). The viscosity of the mucus increases from the time of insemination and throughout pregnancy (Lopez-Gatius et al., 1993; Lopez-Gatius et al., 1994). Stimulation with luteinizing hormone (LH) also induces changes in the endometrium during pro-estrus, resulting in a distinct distribution of cells. The main purpose of these modifications is to protect the fetus and the uterus from external environment (El-Banna and Hafez, 1972; Hawk, 1987; Tsiligianni et al., 2003). However, the exact nature of these changes has not been reported in detail. In the present study, we found that the cytological profile of vaginal mucus undergoes significant changes during the progression from estrus to gestation. We observed large variations in the viscosity and physiological properties of vaginal mucus at the time of insemination and during pregnancy. Our results highlight the role played by cytological and physiological properties of vaginal mucus in reproduction, which have been widely recognized during the ovarian cycle where hormonal imbalance results in abnormal regulation (El-Banna and Hafez, 1972; Eliezer, 1974; Tam et al., 1980; Carlstedt and Sheehan, 1989; Lopez-Gatius et al., 1993; Lopez-Gatius et al., 1997).

 Hormonal changes during the estrus cycle induce differential expression of vaginal mucus-specific proteins to help the movement of spermatozoa (Chung et al., 2008). To identify proteins undergoing this change, we performed mass spectrometry of 6 proteins selected from the 16 proteins detected in the vaginal mucus. Among these, 5 were previously identified by Chung et al. (2008). The sixth, MMP-9 plays an important role in tissue remodeling in various physiological processes, including implantation and ovarian and uterine functions during estrus and pregnancy (Smith, 1999; Woessner, 2002).

 One of the proteins we detected from the vaginal mucus in estrus and gestation is MMP-9. MMPs are proteolytic enzymes that depend on zinc and calcium ions and are the main factors causing ECM degradation in various tissues (Nagase and Woessner, 1999). Detection of MMP-9 in the vaginal mucus is meaningful because endometrial remodeling is essential for successful implantation (Blankenship and King, 1994; Huppertz et al., 1998; Salamonsen, 1999; Bjorn et al., 2000; Qin et al., 2003). Therefore, MMP-9 can be employed as an indirect probe of the state of the endometrium during estrus and early pregnancy. To confirm the expression of MMP-9, we performed gelatinase zymography and ELISA with vaginal mucus samples. We found that MMP-9 activity in vaginal mucus is highest on estrus day 0 and pregnancy day 210. It appears to me that the ELISA and zymography results both show the MMP-9 levels are high at estrus day 0 and stay more or less stable through pregnancy. These expression patterns match those earlier observed in the endometrium during estrus and pregnancy (Guillomot, 1999; Kizaki et al., 2008).

 In summary, we have identified different characteristics in vaginal mucus from estrus and pregnancy. We also detected differences in MMP-9 expression between estrus and pregnancy. Our results indicate that the gelatinase B activity of MMP-9 increases in response to the state of the endometrium, and may be useful in predicting the time of estrus. The development of such an indicator would be useful for the improvement of conception by the application of artificial insemination at the optimal time. 


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