Major proteins of bovine seminal plasma and high-density lipoprotein induce cholesterol efflux from epididymal sperm

One of the hypotheses to explain the mechanism of capacitation involves the loss of sperm membrane cholesterol. Here, we studied whether or not the major proteins of bovine seminal plasma designated as BSP-A1, -A2, -A3, and -30-kDa (collectively called BSP proteins), which are implicated in sperm capacitation, induce cholesterol efflux. When epididymal sperm were labeled with [3H]cholesterol and incubated with bovine seminal plasma (0.05-2%) or BSP proteins (20-120 microg/ml) for 8 h, the sperm lost [3H]cholesterol (3.6-fold and 3-fold, respectively). The same results in the presence of BSP-A1/-A2 were obtained (3.5-fold) by direct determination of cholesterol on unlabeled epididymal sperm. Analysis of efflux particles by ultracentrifugation on a sucrose gradient revealed a single symmetrical peak of radioactivity at 1.14 g/ml. Immunoblotting of the fractions obtained from size-exclusion chromatography of the efflux particles showed that a portion of the BSP proteins were associated with [3H]cholesterol. Heparin (12 microg/ml) alone did not stimulate cholesterol efflux. In contrast, high-density lipoprotein (HDL, 100 microg/ml) alone stimulated cholesterol efflux up to 3.1-fold after 8 h. When labeled epididymal sperm were preincubated for 20 min with BSP-A1/-A2 (120 microg/ml), washed, and incubated with HDL (100 microg/ml) for 8 h, the total cholesterol efflux of the sperm suspension was 51.8 +/- 5.0% compared to 39.3 +/- 1.2% when HDL alone was used. These results indicate that BSP proteins and HDL play an important role in the sperm sterol efflux that occurs during capacitation. Furthermore, the heparin-induced sperm capacitation did not involve the efflux of sperm membrane cholesterol.     

Combined positive/negative purging and transplantation of peripheral blood progenitor cell autografts in breast cancer patients: a pilot study

Capacitation is an important process in bovine sperm maturation and is an obligatory step prior to fertilization. Two capacitating agents, namely heparin and high-density lipoprotein (HDL), have been shown to induce sperm capacitation. A family of major proteins of bovine seminal plasma designated BSP-A1/A2, BSP-A3, and BSP-30 kDa (collectively called BSP proteins) bind to the sperm surface upon ejaculation via their membrane choline phospholipids. Our previous studies with bovine epididymal sperm showed that BSP proteins potentiate sperm capacitation induced by heparin and HDL. This study was undertaken to clarify the mechanism of capacitation induced by heparin and HDL in the presence of BSP proteins. Washed bovine ejaculated sperm were incubated with heparin (12 microg/ml) or HDL (10-160 microg/ml) in the presence of polyclonal antibodies against purified BSP proteins (anti-BSP proteins). The percentage of capacitated sperm was evaluated after the induction of the acrosome reaction (AR) with lysophosphatidylcholine. When sperm were incubated for 5 h with heparin and anti-BSP proteins (40 microg/ml), the AR level was not significantly different from control levels (16. 8 +/- 0.9% vs. 12.9 +/- 0.9%). In contrast, incubation of sperm for 8 h with HDL and anti-BSP proteins did not inhibit the AR (42.4 +/- 1.1% vs. 17.1 +/- 1.6 for the control samples). We also investigated the effect of heparin and HDL on protein tyrosine phosphorylation associated with capacitation. The tyrosine phosphorylation of a group of proteins was increased in the presence of heparin. However, HDL did not significantly stimulate protein phosphorylation. The increase in phosphorylation was correlated with an increase in the AR after the incubation with heparin but not with HDL. These results indicate that heparin and HDL mediate capacitation via different mechanisms.     

Major proteins of bovine seminal plasma modulate sperm capacitation by high-density lipoprotein

Bovine seminal plasma (BSP) contains four similar proteins secreted by the seminal vesicles, designated BSP-A1, -A2, -A3, and -30 kDa. These proteins bind to choline phospholipids on the surface of the sperm after ejaculation. These BSP proteins also interact with heparin, apolipoprotein A-I (apoA-I) and apoA-I associated with high-density lipoprotein (HDL). The HDL and heparin present in the female reproductive tract have been implicated in sperm capacitation and the acrosome reaction (AR). This study was undertaken to determine whether or not these BSP proteins and HDL could modulate the capacitation of sperm, and to determine the combined effect of HDL and heparin on capacitation. Washed bovine epididymal sperm were preincubated in buffer containing BSP proteins, washed, and incubated with lipoproteins (HDL, and low- and very low-density lipoproteins) or liposomes with or without apoA-I in the presence or absence of heparin. The percentage of capacitated sperm was evaluated after the AR was induced with lysophosphatidylcholine. HDL alone (160 microg/ml) after an 8-h incubation stimulated the AR of epididymal sperm. The percentage of HDL-enhanced AR further increased when sperm were preincubated with BSP proteins. ApoA-I-liposomes stimulated the AR more rapidly (5 h, 160 microg/ml) than HDL. When sperm were preincubated with BSP proteins, the percentage of apoA-I-enhanced AR further increased. In contrast, when liposomes without apoA-I or when low- or very low-density lipoproteins or lipoprotein-depleted serum was used, no significant increase in the AR was detected with or without BSP proteins. When heparin and HDL or apoA-I-liposomes were used together, their combined effects on the AR were not additive. These results indicate that BSP proteins modulate the process of capacitation induced by heparin, HDL, and apoA-I-liposomes.     

The hemagglutination-positive phenotype of Mycoplasma synoviae induces experimental infectious synovitis in chickens more frequently than does the hemagglutination-negative phenotype

Inoculation with hemagglutination-positive (HA+) cultures of Mycoplasma synoviae AAY-4 induced acute synovitis significantly more frequently (P = 0.001) in chicken tibiotarsal-tarsometatarsal joints than did inoculation with HA-negative (HA-) cultures derived from the same clone of AAY-4. Immunoblotting analyses showed that HA+ cultures abundantly expressed two phase-variable hemadherence-associated surface membrane proteins of 53 kDa and 48 to 50 kDa defined by monoclonal antibodies. HA- cultures lacked the 53-kDa proteins and synthesized truncated 27- to 30-kDa forms of the 48- to 50-kDa proteins. Inoculation of cyclosporin A (CsA) into infected joints significantly decreased the frequency of acute synovitis (P = 0.001). Moreover, repeated intra-articular inoculation of CsA (three doses of 1 mg at 2-day intervals) significantly reduced the local antibody response to M. synoviae in the joints treated with CsA.     

Purification and partial characterization of candidate antidiuretic hormone water channel proteins of M(r) 55,000 and 53,000 from toad urinary bladder

Antidiuretic hormone (ADH) increases toad bladder granular cell apical membrane osmotic water permeability (Pf) by insertion of cytoplasmic vesicles containing water channels into the apical membrane. Termination of ADH stimulation results in endocytosis of water channel-containing membrane. In previous work, we have purified water channel-containing vesicles and demonstrated that they contain 12 major protein bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of vectorial labeling studies of granular cells and purified vesicles, we have proposed previously that vesicle proteins of 55, 53, and 17 kDa are ADH water channel components. In this report, we have purified and analyzed these three proteins using a combination of SDS-PAGE, peptide mapping, amino acid composition, and amino-terminal analyses. The 55- and 53-kDa proteins are distinct protein species possessing a high degree of structural similarity. Both possess a large content of cysteine. The 17-kDa protein appears to be a proteolytic fragment of the 53-kDa protein. None of these three proteins is phosphorylated or contains large amounts of covalently linked carbohydrate. ADH-elicited Pf is inhibited by the organic mercurial reagent fluorescein mercuric acetate (FMA). Exposure of water channel-containing vesicles to FMA labels selectively four vesicle proteins of 92, 55, 53, and 29 kDa while reducing vesicle Pf by 82%. The combination of FMA and 2-mercaptoethanol or exposure to another mercurial reagent, n-ethylmaleimide, does not inhibit vesicle Pf. Together, these data provide additional evidence for the role of the 55- and 53-kDa proteins as components of the ADH water channel. These candidate ADH water channel proteins are distinct from a 28-kDa candidate water channel protein (CHIP 28) isolated recently from human erythrocyte membranes and kidney proximal tubule by Agre and co-workers (Preston, G. M., Carroll, T. P., Guggino, W. B., and Agre, P. (1992) Science 256, 385-387).     

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.     

Osteopontin is the 55-kilodalton fertility-associated protein in Holstein bull seminal plasma

Previously we identified four proteins in seminal plasma that were associated with bull fertility. The purpose of this study was to identify the 55-kDa protein prevalent in seminal plasma of higher-fertility males. The 55-kDa protein was quantified by video densitometry in two-dimensional electrophoresis gels of seminal plasma from 26 bulls of known fertility. Relative density of the 55-kDa protein was positively correlated (r = 0.48) with bull fertility. The 55-kDa (pI 4.5) fertility-associated protein spot was isolated by electroelution after two-dimensional PAGE separation of seminal plasma of 36 bulls. N-terminal sequence analysis of the pure protein yielded a 15-amino acid sequence (VKPXSSGXSEEKQLN) that was 86% homologous to bovine osteopontin-k precursor. Polyclonal antiserum generated against the 55-kDa protein reacted with a single spot in two-dimensional PAGE Western blots of seminal plasma. Western blot analyses using polyclonal antisera generated against the amino terminus (LF123) and carboxyl terminus (LF124) of human recombinant osteopontin confirmed that the 55-kDa polypeptide was osteopontin. Partially purified 55-kDa protein was obtained by HPLC-MonoQ column chromatography. Protein characterization revealed that the 55-kDa protein was glycosylated, but not phosphorylated, consistent with the identity of the 55-kDa protein as osteopontin.     

Influence of antimicrobial chemotherapy on spirometric parameters and pro-inflammatory indices in severe pulmonary tuberculosis

Several proteins, which are recognized components of serum, are not resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) under standard conditions. One major example is fibronectin, which is detected in fairly high concentration (milligram range) by immunoassays, while undetectable in 2D-PAGE gels. Following several experiments with a combination of zwitterionic and chaotropic substances we obtained a good resolution of the protein in gels containing 0.5 M thiourea plus 8 M urea. By this technique, fibronectin was, for the first time, found to be microheterogeneous between pl values of 5.3 and 5.6. Besides fibronectin we detected three other families of uncharacterized proteins with Mr of 130000, 110000 and 34000 respectively, whose identity and function are currently under investigation.     

Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung

A protein (mCLCA1) has been cloned from a mouse lung cDNA library that bears strong sequence homology with the recently described bovine tracheal, Ca2+-sensitive chloride channel protein (bCLCA1), bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1), and the human intestinal Ca2+-sensitive chloride channel protein (hCLCA1). In vitro, its 3.1-kilobase message translates into a 100-kDa protein that can be glycosylated to an approximately 125-kDa product. SDS-polyacrylamide gel electrophoresis from lysates of mCLCA1 cDNA-transfected transformed human embryonic kidney cells (HEK293) reveals proteins of 130, 125, and 90 kDa as well as a protein triplet in the 32-38 kDa size range. Western analyses with antisera raised against Lu-ECAM-1 peptides show that the N-terminal region of the predicted open reading frame is present only in the larger size proteins (i.e. 130, 125, and 90 kDa), whereas the C-terminal region of the open reading frame is observed in the 32-38 kDa size proteins, suggesting a posttranslational, proteolytic processing of a precursor protein (125/130 kDa) into 90 kDa and 32-38 kDa components similar to that reported for Lu-ECAM-1. Hydrophobicity analyses predict four transmembrane domains for the 90-kDa protein. The mCLCA1 mRNA is readily detected by Northern analysis and by in situ hybridization in the respiratory epithelia of trachea and bronchi. Transient expression of mCLCA1 in HEK293 cells was associated with an increase in whole cell Cl- current that could be activated by Ca2+ and ionomycin and inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, dithiothreitol, and niflumic acid. The discovery of mCLCA1 opens the door for further investigating the possible contribution of a Ca2+-sensitive chloride conductance to the pathogenesis of cystic fibrosis.     

Purification and characterization of the acid soluble 26-kDa polypeptide from soybean seeds

Whey proteins from soybean seeds of Japanese varieties were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Among 11 varieties of soybean, three green and one black soybeans lacked a 26-kDa band that was found in all yellow soybeans. In this paper, the 26-kDa protein was named AS26k (acid soluble 26-kDa protein) temporarily. The AS26k protein was purified from Glycine max cv. Nattosyoryu, which is yellow soybean, through four purification steps: 30-35% saturated ammonium sulfate fractionation, ion exchange chromatography on S Sepharose Fast Flow, gel filtration on Sephadex G-100, and hydrophobic chromatography on phenyl Sepharose CL-4B. Purified AS26k was cleaved with V8 proteinase from Staphylococcus aureus or CNBr. The cleaved polypeptide contained two typical dehydrin motif sequences: DEYGNPV and (M)DKIKEKLPG, and a 19 amino acids sequence similar to a pea dehydrin. Native AS26k had a molecular mass of 32 kDa on gel filtration and a pl of 7.2 on two-dimensional PAGE. Similarly to other dehydrins and late embryogenesis abundant (LEA) proteins, AS26k was rich in hydrophilic amino acids, and highly heat stable. These results showed that AS26k was a dehydrin, a group II LEA protein in soybean seeds.     

Protein kinases of cultured chicken osteoblasts that phosphorylate extracellular bone proteins

Cytosolic and microsomal protein kinase preparations from cultured chicken osteoblasts were found to phosphorylate up to six major proteins with Mrs 66, 58, 50, 36, 32, and 22 kDa in chicken bone extract. Use of heparin led to the conclusion that these proteins were predominantly phosphorylated by factor-independent protein kinase (FIPK) present both in microsomal and cytosolic preparations. It was confirmed that microsomal preparation contained predominantly FIPK, whereas cytosolic preparation contained additional kinases, that can phosphorylate the bone proteins. Use of purified chicken bone osteopontin (OPN) (58 kDa) and recombinant OPN led to the same conclusions. The identify of the protein kinases was clearly established by using a series of synthetic peptide substrates. Quantitative analysis utilizing pure protein kinases and purified chicken bone OPN, recombinant mouse OPN, and bovine bone OPN and BSP led to introduction of approximately 9 moles of phosphate/mole of OPN and 6.6 moles phosphate/mole bovine bone sialoprotein (BSP) by casein kinase II. cGMP-dependent protein kinase and protein kinase C both introduced 0.5-1.2 moles phosphate/mole of OPN and BSP, whereas cAMP-dependent protein kinase led to no significant phosphorylation of OPN or BSP. Consistent with the above results, sites of phosphorylation identified for OPN (metabolically labeled) and BSP (labeled by casein kinase II) revealed that predominant phosphorylated sites have recognition sequences for FIPK.     

Protein phosphatase 2A catalytic subunit is methyl-esterified at its carboxyl terminus by a novel methyltransferase

In eukaryotic cells a number of different proteins with important regulatory functions are reversibly methyl-esterified at carboxyl-terminal prenylcysteine residues. These proteins include the low molecular weight GTP-binding proteins, the gamma-subunit of the heterotrimeric G-proteins, and the nuclear lamins. The methylating enzymes that catalyze this type of carboxyl methylation reaction are integral membrane proteins, and the methylated protein products tend to be membrane-associated. Analyses of protein carboxyl methylation in a wide range of vertebrate tissues revealed a major carboxyl-methylated protein that was clearly distinct from those that are modified at prenylcysteine groups (Volker, C., Miller, R.A., McCleary, W.R., Rao, A., Poenie, M., Backer, J.M., and Stock, J.B. (1991) J. Biol. Chem. 266, 21515-21522). This M(r) = 36,000 protein is localized to the cytosol. Unlike the prenylcysteine methyltransferases, the enzyme that catalyzes the methylation of the 36-kDa protein is found in the cytosol. The 36-kDa methylated protein has been purified from bovine brain. Sequence analysis of several peptides clearly shows that the protein is the catalytic subunit of protein phosphatase 2A. A soluble 40-kDa methyltransferase that catalyzes the reaction has also been purified.     

Expression of a recombinant human growth hormone binding protein in baculovirus/insect cell system

An eucaryotic recombinant human growth hormone binding protein (rGHBP) was expressed in baculovirus-infected insect cells and purified by affinity chromatography from culture supernatant. This mannose-rich 34-kDa protein specifically bound human growth hormone (hGH) with the same affinity (kDa = 0.42 x 10(-9) M) than the 51.5 kDa GHBP we purified and characterised from human plasma (kDa = 1.1 x 10(-9) M). A high molecular form of the rGHBP was detected by silver-stained SDS-PAGE, Western blot (mAb 263), affinity cross-linking and Western ligand blot with 125I-hGH. Reduction experiments with beta-mercaptoethanol suggested that this form involved a disulfide bound between two rGHBPs.     

Purification of a bone sialoprotein-binding protein from Staphylococcus aureus

Bone sialoprotein (BSP) is selectively bound by Staphylococcus aureus cells isolated from patients suffering from infections of bone and joint tissues [Rydén C., Maxe, I., Franzén, A., Ljungh, A., Heineg?rd, D. & Rubin, K. (1987) Lancet II, 515]. We now report on the purification of a cell-wall protein from Staphylococcus aureus, strain O24, that possesses affinity for bone sialoprotein. Staphylococcal cell-wall components with capacity to inhibit binding of 125I-labeled BSP to staphylococcal cells were solubilized with LiCl (1.0 M, pH 5.0). Preparative SDS/PAGE and protein-overlay experiments revealed that inhibitory activity present in LiCl extracts resided in a fraction of polypeptides with M(r) 75,000-110,000. Staphylococcal proteins solubilized with LiCl were chromatographed on a Mono-Q anion-exchange column. Inhibitory activity was eluted at 0.6-0.8 M NaCl and could be further purified by affinity chromatography on BSP-Sepharose. Elution of the affinity matrix with 0.1 M glycine, pH 3.0, specifically eluted inhibitory activity. Analysis by SDS/PAGE revealed a single M(r) 97,000 polypeptide in the eluate. The purified M(r) 97,000 protein bound BSP in protein-overlay experiments. LiCl extracts from S. aureus, strain E514 or Staphylococcus epidermidis, strain 7686, both lacking the capacity to bind BSP did not contain the M9r) 97,000 protein. Our data demonstrate the presence of a S. aureus cell-surface BSP-binding protein. This protein could be involved in bacterial tropism in osteomyelitis.     

Ubiquitin is conjugated to the cytoskeletal protein alpha-spectrin in mature erythrocytes

Ubiquitination of red blood cell (RBC) proteins was investigated by encapsulation of 125I-ubiquitin into human erythrocytes using a procedure of hypotonic dialysis, isotonic resealing, and reannealing. Incubation (37 degrees C, up to 2 h) of 125I-ubiquitin-loaded cells resulted in the recovery of 125I-ubiquitin with the cytosolic proteins (9.22 +/- 0.4 micrograms/ml RBC) and conjugated to membrane proteins (2.18 +/- 0.05 micrograms/ml RBC). This conjugation was time-dependent, and the predominant membrane protein band that became labeled showed an apparent molecular mass of 240 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Western blotting experiments with three different anti-ubiquitin antibodies revealed that this protein is also ubiquitinated in vivo. Cell-free experiments have shown that fraction II (a DEAE-bound protein fraction eluted by 0.5 M KCl) prepared from both mature erythrocytes and reticulocytes is able to conjugate ubiquitin to this protein. Ubiquitin conjugation was ATP-dependent (Km 0.09 mM), time-dependent, and fraction II-dependent (8 +/- 0.5 pmol of 125I-ubiquitin/h/mg of fraction II). Isolation of the major RBC membrane protein that is ubiquitinated was obtained by using biotinylated ubiquitin. Membrane proteins, once ubiquitinated with this derivative, were extracted and purified by affinity chromatography on immobilized avidin. The major components retained by the column were two peptides of molecular masses 220 and 240 kDa. Both proteins are recognized by a monoclonal anti-spectrin antibody, but only the 240-kDa component is detected by streptavidin peroxidase conjugate. That indeed the ubiquitinated membrane protein of 240-kDa is alpha-spectrin was confirmed by immunoaffinity chromatography using 125I-ubiquitin and a monoclonal anti-spectrin antibody. Antigen-antibody complexes were purified by protein A chromatography and analyzed by SDS-PAGE and autoradiography. Again two bands of 240 and 220 kDa were eluted (alpha- and beta-spectrin), but only one band corresponding to the electrophoretic mobility of alpha-spectrin was detected by autoradiography. Thus, alpha-spectrin is a substrate for the ATP-dependent ubiquitination system, suggesting that the cytoskeleton is covalently modified by ubiquitination both in reticulocytes and mature RBC.     

Amino acid sequence analysis of the proteolytic cleavage products of the bovine immunodeficiency virus Gag precursor polypeptide

Bovine immunodeficiency virus Gag proteins were purified from virions, and their amino acid sequences and molecular masses were determined. The matrix, capsid, and nucleocapsid (MA, CA, and NC, respectively) and three smaller proteins (p2L, p3, and p2) were found to have molecular masses of 14.6, 24.6, and 7.3 and 2.5, 2.7, and 1.9 kDa, respectively. The order of these six proteins in the Gag precursor, Pr53gag, is NH2-MA-p2L-CA-p3-NC-p2-COOH. In contrast to other retroviral MA proteins, the bovine immunodeficiency virus MA retains its N-terminal methionine and is not modified by fatty acids. In addition, the bovine immunodeficiency virus NC migrates as a 13-kDa protein in denaturing gel electrophoresis; however, its molecular mass was determined to be 7.3 kDa.     

Intercellular interactions as a basis for the expedient behaviour of multicellular systems

Human seminal plasma sperm motility inhibitor (SPMI) proteins which are exclusively secreted from seminal vesicles, inhibit sperm motility. It is secreted as biologically active 52 kDa and a mixture of 71 and 76 kDa precursor forms, which are identical to semenogelin-I and II (Sg-I and Sg-II), respectively. To understand the molecular mechanism underlying the inhibition of sperm motility by SPMI proteins, we expressed human Sg-I and Sg-II genes in insect cells using a baculovirus system. The baculoviruses expressing full-size Sg-I and Sg-II proteins that were N-terminally-tagged with a hexahistidine were selected, and were infected with Sf 21 cells. The Sg-I and Sg-II proteins were purified from infected cells by column chromatography using Ni-NTA resin 48 h after infection. The full-size Sg-I and Sg-II proteins were obtained in soluble forms. However, they tended to aggregate to form a gel, as expected from naturally occurring semenogelin. Both the purified recombinant Sg-I and Sg-II proteins showed strong SPMI activities with a complete inhibition of sperm motility at 60 units/mg, equivalent to the natural proteins. This production system that permits the generation of purified Sg-I and Sg-II proteins, as well as mutant derivatives, will be helpful for further study on male infertility.     

Identification of matrix metalloproteinases and metalloproteinase inhibitors in bovine corpora lutea and their variation during the estrous cycle

Corpora lutea (CL) were collected from cattle to study key physiologic events in angiogenesis. Our objective was to evaluate the activity of matrix metalloproteinases (MMP) and endogenous inhibitors. Corpora lutea were collected 2, 4, 6, 8, 10, 12, 14, and 16 d (n = 3/d) after estrus was first detected. In zymograms, a band of protein migrating at a relative molecular mass (M(r)) of 98 kDa was increased early in the cycle; a M(r) = 88 kDa band was detectable on all days. The molecular weights of these proteins are consistent with the MMP-9 family members. In all samples, a band of enzyme activity was detected at M(r) = 62 kDa, and another band of lesser density was detected at M(r) = 60 kDa. The molecular weights of these proteins are consistent with the MMP-2 family members. An immunoreactive band, detected in all samples with equal density, migrated between M(r) = 27 and 29 kDa, as did the tissue inhibitor of metalloproteinase (TIMP-1) standard. A second band, which was less dense in samples from d 2 through 6, migrated at M(r) = 19 kDa, as did the TIMP-2 standard. A third band was detected in all samples; it migrated at M(r) = 35 kDa, as did the cartilage-derived inhibitor (CDI) standard, and was less dense in d 8 and d 12 through 16 samples. In summary, MMP (gelatinases) and MMP inhibitors are present in developing luteal tissue, and the M(r) = 98 kDa enzyme, CDI, and TIMP-2 varied during the estrous cycle.