Gelatinases in Boar Seminal Plasma and Their Relation to Semen Indicators

Matrix metalloproteinases were detected in reproductive tissues and seminal plasma of various animal species. The aim of this study was to determine for the first time the presence of gelatinases and metalloproteases in boar seminal plasma and to correlate the results with semen indicators. Gelatin zymography was used for simultaneous identification and measurement of gelatinase enzyme activity associated with their molecular weights. Several gelatinase forms were identified in seminal plasma of boars. Those that were stimulated by CaCl2 and inhibited by EDTA and phenanthroline were considered as metalloproteases. Negative correlation between semen indicators (sperm index, sperm concentration and concentration of progressive motile sperm) and the concentrations of metalloprotease at 78 kDa and 66 kDa means that higher values of semen indicators correlate with lower concentrations of these metaloproteases in seminal plasma. Gelatinases with molecular weight of 225, 78 and 66 kDa correlated with higher levels of acrosome damage. Samples with sperm index above 110 M/ml contained gelatinases of significantly lower band intensities at 78 and 66 kDa compared to samples with SI less than 110 M/ml. Bands with 225, 78 and 66 kDa are suggested to belong to a dimer of MMP-9, proMMP-2 and mature MMP-2. Matrix metalloproteinases, boar semen evaluation, semen characteristics Numerous proteolytic enzymes and their inhibitors are localized in both spermatozoa and seminal plasma in the mammalian semen. The spermatozoa are rich in different proteases, which are mainly localized within the acrosomal vesicle (Tulsiani et al. 1998). Seminal plasma contains many proteinases originating either from testicular cells or prostate and other accessory sex glands. Most proteinases described for seminal plasma are serine proteinases, however, proteinases from other classes are present in semen from human and domestic mammals (Metayer et al. 2002). The fertilization process requires breakdown of the physiological barrier during sperm penetration through the zona pellucida and the egg plasma membrane, but a detailed molecular mechanism is still unclear. It is suggested that matrix metalloproteinases (MMPs) can be involved in this process (Buchman-Shaked et al. 2002). The MMPs are a family of proteolytic enzymes capable of degrading specific components of extracellular matrix (ECM) at physiological pH in a zinc-dependent manner. MMPs and their tissue inhibitors (TIMPs) play a key role in many physiological processes, including ovulation and implantation. Although little is known about MMP expression and their exact function in the male reproductive tract, they are believed to be involved in the regulation of spermatozoal function (Hulboy et al. 1997). Matrix-metalloproteinases (MMPs) are zinc-dependent proteinases involved in tissue remodelling, cell migration, and cell-cell interaction (Robinson et al. 2001), and are known as the main enzymes digesting the extracellular matrix. MMPs are secreted in inactive forms that are activated by cleavage of the inhibitory pro-peptide of about 10 kDa (Nagase and Woessner 1999). Keeping sperm proteases in an inactive form is critical for maintaining cell integrity in order to ensure reproductive function of sperm cells (Zheng et al. 1994). A complex mechanism controlling MMP activation includes regulation at the level of gene expression, cleavage of inactive forms and inhibition of active MMPs ACTA VET. BRNO 2010, 79: 491–496; doi:10.2754/avb201079030491 Address for correspondence: Petra Zrimšek Clinic for Reproduction and Horses Veterinary Faculty, University of Ljubljana Gerbičeva 60, Ljubljana, Slovenia Phone: +386 14 779 271 Fax: +386 12 832 243 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm by endogenous inhibitors, primarily tissue inhibitors of MMPs (Nagase and Woessner 1999). Gelatinases have been detected in epididymal fluids of ram, stallion and boar (Metayer et al. 2002). Metalloproteases, serine proteinases and serine proteinase inhibitors have been detected in turkey seminal plasma (Kotlowska et al. 2005). Complexes of gelatinases and TIMP-1 and TIMP-2 (Shimokawa et al. 2003) or MMP-2 and MMP-9 (Shimokawa et al. 2002) were found in human seminal plasma. The first report of gelatinase in human sperms indicated higher 28-kDa gelatinase activity and lower 92-kDa MMP activity in normal compared to abnormal semen (Buchman-Shaked et al. 2002). TIMP-2 in bovine seminal plasma has been described as a factor influencing the bull fertility (McCauley et al. 2001). The role of TIMPs for human sperm function is most probably based on interaction with MMPs in spermatozoa (Baumgart et al. 2002). MMP-2 and MMP-9 were evaluated in human seminal plasma, where latent forms of both MMPs predominate, and pro-MMP-9 was detected in samples of low sperm concentration (Tentes et al. 2007). The aim of this study was to examine the presence of gelatinases and metalloproteases in boar seminal plasma. Materials and Methods Semen samples and their analysis Twenty-one semen samples were obtained from 12 to 24-month-old boars of various breeds. While the boar mounted a dummy sow, semen was collected with gloved hand using a clean semen collecting flask that filters gel, dust and bristles out. Semen was kept at the room temperature and analysed within 1 h. Computer-assisted semen analysis (Hamilton Thorne IVOS 10.2; Hamilton Thorne Research, MA, USA) was performed with a Makler counting chamber (Sefi Medical Instruments, Israel) to determine sperm concentration and motility characteristics. Sperm morphology was examined in Giemsa-stained samples (Hafez 1993). Metabolic activity of spermatozoa was assayed using a spectrophotometric application of the resazurin reduction assay (Zrimšek et al. 2004; Zrimšek et al. 2006). Sperm index was calculated by multiplying the total sperm concentration by the square root of its motility multiplied by the percentage of normal sperm (Mahmoud et al. 1994). Seminal plasma preparation Samples of seminal plasma were prepared at the same time as evaluation of the semen. Following centrifugation at 818 g for 10 min at room temperature, the supernatant was further centrifuged at 13,000 g for 15 min at 4 oC to separate seminal plasma, which was then aliquoted and frozen at –80 oC until assayed. Gelatin zymography Gelatin zymography was performed on seminal plasma using a modified method of Laemmli (1970). The 7.5% separating polyacrylamide gel (0.75 mm thickness) contained 0.12% pig skin type I gelatin (Sigma, Germany), whereas the 4% stacking gel contained no gelatin. The samples of seminal plasma, diluted 1:5 in PBS, were denatured with SDS but not reduced. The mixture of molecular weight markers was diluted 1:4 with SDS reducing buffer and heated in a boiling water-bath for 5 min prior to loading on the gel. MMP-2 from human fibroblasts (Sigma) and MMP-9 from human fibroblasts (Sigma) were used as controls. Each sample of 10 μl was loaded into a well and samples were electrophoresed at a constant 200 V for 45 min. After electrophoresis, the proteins were allowed to renature by removing SDS by washing the gel in 2.5% Triton X-100 with gentle shaking for 30 min at room temperature. Gels were incubated overnight at 37 oC in a reactivation buffer of pH 7.6 containing 50 mM Tris/HCl, 10 mM CaCl2, 150 mM NaCl and 0.2% Brij 35. Following incubation, the gels were stained for 20 min with 0.1% Coomassie brilliant blue R-250 (Bio-Rad, Germany) in a solvent mixture containing 40% methanol and 10% acetic acid. After staining, the gels were de-stained in the same solution in the absence of dye until gelatinolytic bands became white against a blue background. De-stained gels were scanned using a Model GS-700 Imaging Densitometer (Bio-Rad). Relative intensities of gelatinolytic bands, representing the gelatinolytic activity, were quantified using GIMP 2.2.10 software. Average pixel intensities of the bands were reduced for the pixel intensity of the gel’s background. To examine the inhibition of enzyme activity, gels were also incubated in reactivation buffer without CaCl2, and reactivation buffer containing 10 mM EDTA or 2 mM o-phenanthroline. Statistical analysis Spearman rank correlation coefficients were calculated to correlate the concentrations of gelatinases with each other and with sperm indicators such as sperm concentration, motile sperm concentration, sperm index, metabolic activity, % of morphological normal spermatozoa and % of spermatozoa with damaged acrosome. In order to investigate the possible association of gelatinases with the above sperm indicators, semen samples were divided into two groups according to a sperm index (SI) of 110 M/ml: group A (SI < 110 M/ml; n = 9) and 492