key: cord-0042781-4guqlzrs
authors: Peltier, Morgan R.; Barney, Brandon M.; Brown, Mary B.
title: Effect of Experimental Genital Mycoplasmosis on Production of Matrix Metalloproteinases in Membranes and Amniotic Fluid of Sprague–Dawley Rats
date: 2007-01-08
journal: Am J Reprod Immunol
DOI: 10.1111/j.1600-0897.2006.00449.x
sha: 33020c2cf6e00ec7bf227a68a5889888e298abe0
doc_id: 42781
cord_uid: 4guqlzrs

PROBLEM: Preterm, premature rupture of membranes (PPROM) is a dire pregnancy outcome that is frequently associated with infection by the genital mycoplasmas, Mycoplasma hominis, Ureaplasma parvum, and U. urealyticum. One potential mechanism by which these microorganisms may cause PPROM is by increasing the concentration of matrix metalloproteinases (MMPs) in the membranes and amniotic fluid. We tested this hypothesis in a well‐defined model system of genital infection with M. pulmonis, a natural reproductive pathogen of rats. METHOD OF STUDY: Timed‐pregnant, specific pathogen‐free, Sprague–Dawley rats were infected with 10(7) CFU M. pulmonis at gestation day (gd) 14. Controls received an equivalent volume (100 μL) of sterile medium. At gd 18, rats were euthanized, and membranes and amniotic fluids were harvested and stored at −70°C until analysis. Proteinase activity of amniotic fluid and membranes was resolved on discontinuous 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gelatin zymography gels. Band intensity was determined using a digital gel documentation system and the manufacturer's software (Kodak). RESULTS: Gelatinolytic activity associated with a band similar in molecular weight to ProMMP‐9 (92 kDa, the inactive precursor of MMP‐9) was significantly increased in amniotic fluids and membranes harvested from M. pulmonis‐treated pups at gd 18 when compared with tissues harvested from control pups. Both ProMMP‐9 and ProMMP‐2 (72 kDa, the inactive precursor of MMP‐2) were increased in infected animals at gd 21. CONCLUSION: Our study suggests that the genital mycoplasmas can increase MMP‐9 production in vivo.

Preterm, premature rupture of membranes (PPROM) is a dire pregnancy outcome that is frequently associated with infection by the genital mycoplasmas, Mycoplasma hominis, Ureaplasma parvum, and U. urealyticum. One potential mechanism by which these microorganisms may cause PPROM is by increasing the concentration of matrix metalloproteinases (MMPs) in the membranes and amniotic fluid. We tested this hypothesis in a well-defined model system of genital infection with M. pulmonis, a natural reproductive pathogen of rats.

Timed-pregnant, specific pathogen-free, Sprague-Dawley rats were infected with 10 7 CFU M. pulmonis at gestation day (gd) 14 . Controls received an equivalent volume (100 lL) of sterile medium. At gd 18, rats were euthanized, and membranes and amniotic fluids were harvested and stored at )70°C until analysis. Proteinase activity of amniotic fluid and membranes was resolved on discontinuous 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gelatin zymography gels. Band intensity was determined using a digital gel documentation system and the manufacturer's software (Kodak).

Gelatinolytic activity associated with a band similar in molecular weight to ProMMP-9 (92 kDa, the inactive precursor of MMP-9) was significantly increased in amniotic fluids and membranes harvested from M. pulmonis-treated pups at gd 18 when compared with tissues harvested from control pups. Both ProMMP-9 and ProMMP-2 (72 kDa, the inactive precursor of MMP-2) were increased in infected animals at gd 21.

Our study suggests that the genital mycoplasmas can increase MMP-9 production in vivo.

cum are often isolated from the reproductive tract of women with this condition. 2, 3 Mycoplasma hominis and Ureaplasma sp. are thought to cause damage to the membranes by increasing the production of proinflammatory cytokines at the maternal-fetal interface through interactions with immune cells or direct interactions with trophoblast cells themselves. Indeed, both M. hominis and U. urealyticum increased the production of tumor necrosis factor-alpha (TNF-a) and nitric oxide by murine macrophages in vitro, 4 and both microorganisms can cause preterm labor when administered by intraamniotic infusion into rhesus monkeys. 5,6 TNF-a and other proinflammatory cytokines can disrupt pregnancy in a number of ways, including upregulation of the production of matrix metalloproteinases (MMPs).

Matrix metalloproteinases comprise a family of over 20 members and can be divided into groups based on their substrates (e.g. stromalysins, gelatinases, collagenases, etc). MMP-9 is particularly important in remodeling of the membranes and cervix during parturition. MMP-9 is upregulated in the membranes at the time of labor in the human, monkey, and rat. [7] [8] [9] This enzyme is also increased in the amnion by group B streptococci 8 or bacterial cell wall components such as lipopolysaccharide (LPS). 10 MMP-2 has also been shown to be increased during preterm rupture of membranes. 11 Whether genital mycoplasmas that lack LPS can increase MMP-9 or MMP-2 production in this tissue is unknown.

Our laboratory has been investigating the pathophysiology of genital mycoplasmosis using Sprague-Dawley rats and M. pulmonis. This animal model system has several advantages for investigations in reproductive pathology because it uses a natural pathogen of rodents that is associated with increased production of proinflammatory cytokines, low birthweight and fetal wastage. 12 In this study, we tested the hypothesis that genital mycoplasmosis causes increased production of MMP-2 and MMP-9 in the membranes and amniotic fluid.

Coomassie Blue R-250 and acrylamide-bis acrylamide solution were purchased from Bio-Rad (Hercules, CA, USA). All other chemical reagents were purchased from either Sigma (St Louis, MO, USA) or Fisher Scientific (Atlanta, GA, USA). Mycoplasma pul-monis strain X1048 used for this project was generously provided by Dr Maureen Davidson (West Lafayette, IN, USA). A stock culture was grown in Frey's broth, stored in aliquots at )85°C, and a single vial from this stock culture was thawed just prior to use. The thawed culture contained 10 8 CFU/mL when thawed, and the CFU was confirmed for each experiment.

All experiments were performed in accordance with University of Florida Institutional Animal Care and Use Committee-approved protocols. Timed-pregnant, specific pathogen-free (SPF) Sprague-Dawley rats were purchased from Harlan (Indianapolis, IN, USA) and delivered to the University of Florida at gestation day (gd) 11 or 12 and maintained under SPF conditions at all times. Rats were monitored by the commercial vendor and were presumed SPF for the following: Sendai virus, H-1 virus, rat coronavirus, sialodacryoadenitis virus, reovirus type 3, Kilham rat virus, Hantaan virus, M. pulmonis, respiratory and enteric pathogens, endoparasites and ectoparasites and housed in MicroisolatorÒ (Lab Products, Inc., Maywood, NJ, USA) cages. In order to maintain SPF conditions, all animals were handled within a laminar airflow hood. All food, water and caging were autoclaved prior to use. Control rats were always handled first and were housed separately from inoculated rats.

On gd 14, rats were anesthetized with an intraperitoneal dose of ketamine (25 mg; Ketaject, Phoenix Pharmaceutical Inc., St Joseph, MO, USA) mixed with xylazine (0.375 mg; Xylazine-20, The Butler Co., Columbus, OH, USA) to produce 40 min of anesthesia. Animals then received either 10 7 CFU M. pulmonis via intracardiac injection, or an equivalent volume of sterile Frey's broth. This method results in nearly a 100% infection rate for the placenta and amniotic fluid by gd 18, 13 severe histological placentitis, and increased production of proinflammatory cytokines at the maternal-fetal interface. 13, 14 At necropsy, gd 18 or 21, rats were anesthetized with an overdose of sodium pentobarbital (180 mg; Veterinary Laboratories Inc., Lenexa, TX, USA) injected intraperitoneally. After deep anesthesia was obtained, rats were exsanguinated by transection of the femoral artery and vein. Fetal rat pups died in utero upon death of the dam. Samples of maternal and fetal tissues were collected to determine the infection status of the rats and their pups. Membranes and amniotic fluid were collected from the fetal units, snap-frozen in liquid nitrogen and stored at )85°C until zymography.

Amniotic fluid was thawed and clarified by centrifugation at 10,000 · g for 15 min at 4°C. The clarified amniotic fluid was mixed 1:3 with zymography loading buffer and loaded onto discontinuous 7.5% (w/ v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels that contained 0.5 mg/mL gelatin. Amniotic fluid obtained at gd 18 was analyzed for MMP-2 (1.6 lL loaded per lane) and for MMP-9 (3.4 lL amniotic fluid loaded per lane). After electrophoresis, gels were incubated in 2.5% Triton X-100 for 1 hr and then incubated in zymography development solution for 18-20 hr at 37°C. Gels were incubated in 0.5% Coomassie Blue in 40% methanol, 7% acetic acid, 53% water R-250 for 1 hr, destained for 1 hr in 40% methanol, 7% acetic acid, 53% water, and photographed using a digital gel analysis system. With this method, bands of proteolytic activity appear as clear bands on a blue background. Band intensities at approximately 72 kDa (ProMMP-2) and 92 kDa (ProMMP-9) were quantified using the Kodak 1D digital gel analysis and documentation system (Rochester, NY, USA).

Membranes were homogenized in 5 mL lysis buffer (50 mm Tris-HCl, 2 m urea, 1 g/L Brij 35, and 0.1 mm phenylmethylsulfonyl fluoride) and the proteins were clarified by centrifugation at 15,000 · g for 15 min. Total protein concentration in the supernatant was measured using the Bio-Rad protein reagent with bovine serum albumin as standard. Protein (100 lg) was then precipitated by adding ice-cold acetone to 90% (v/v) and separated by centrifugation at 10,000 · g for 15 min at 4°C. Samples were then prepared by resuspending the proteins in loading buffer, and 6 lL (30 lg) was analyzed by zymography as described above.

Membranes were homogenized in 1 mL Trizol reagent (Gibco, Grand Island, NY, USA) and total RNA was extracted and precipitated according to the manufacturer's instructions. Complementary cDNA was prepared from 2 lg total RNA using a cDNA archive kit (Applied Biosystems International, Foster City, CA, USA). Real-time polymerase chain reaction (PCR) was performed in duplicate reactions on 2 lL cDNA using master-mix, PCR primers and probes for MMP-9 and 18S rRNA using the TaqMan TM system and associated software as directed by the manufacturer (Applied Biosystems, Foster City, CA, USA).

Potential differences in ProMMP-9 and ProMMP-2 band intensity were evaluated by generalized estimating equations using the generalized linear models procedure of sas (SAS Institute, Cary, NC, USA). Data for MMP-9 gene expression (cycles to amplification) were analyzed in a similar manner except that 18S rRNA was used as a covariate to correct for potential differences in RNA loading. Differences were considered significant at the P < 0.05 level, and data are shown as least-squares mean ± S.E.M.

Culture results for the samples used for these analyses are shown in Table I . Level of infection was gen- erally higher for amniotic fluid and placenta than for the spleen-liver. Gelatinolytic activity was consistently observed at 92 and 72 kDa, corresponding to ProMMP-9 and ProMMP-2. The gelatinolytic activity of these bands could be inhibited by addition of 10 mm ethylenediaminetetraacetic acid to the development solution suggesting that the bands were MMPs (data not shown). Significantly more activity (band intensity) was observed in amniotic fluid (Fig. 1) and membranes (Fig. 2) for the band corresponding to ProMMP-9 in animals that received M. pulmonis than in control animals at gd 18. Real-time reverse transcription (RT)-PCR demonstrated that fewer cycles of amplification were required for MMP-9 in the membranes collected from control animals than in tissues harvested from infected animals (Fig. 3) . No significant differences were detected for gelatinolytic activity for the band corresponding to ProMMP-2 in amniotic fluid or membranes at gd 18. At gd 21, however, more activity for both ProMMP-9 and ProMMP-2 was present in membranes of M. pulmonis-infected dams than control dams (Fig. 4) . 

Preterm premature rupture of membranes accompanies about 50% of all preterm deliveries and is frequently associated with infection. 15 The genital mycoplamas M. hominis and Ureaplasma sp. are most common pathogens of the reproductive tract. These organisms have been isolated from amniotic fluid harvested from women with intact membranes. 16 It has been reported that vaginal colonization or amniotic infection with this organism significantly increases the likelihood of PPROM. 2, 3 Infection with Ureaplasma sp. in women is associated with increased concentrations of TNF-a in amniotic fluid 16 and experimental infection of Rhesus monkeys with either U. urealyticum or M. hominis increases the production of TNF-a in amniotic fluid. 5, 6 This cytokine has been shown in vitro to stimulate MMP-9 production by amnion 10 or amniochorion. 17 Mycoplasma pulmonis has previously been shown to increase TNF-a concentrations in amniotic fluid in experimentally infected rats. 12 Therefore, we hypothesized that genital mycoplasmas would increase in vivo production of MMP-9 in a welldefined system of intra-amniotic infection.

We found that MMP-9 was significantly increased in the membranes and amniotic fluids of M. pulmonis-infected dams. Although M. pulmonis is a different species than the genital pathogens affecting humans (U. parvum, U. urealyticum, M. genitalium, and M. hominis), there are many similarities in the pathophysiology of these organisms with regard to their consequences on infertility and fetal wastage. 12 Although no overt clinical effects of infection on pregnancy are observed with this model, intravenous injection of M. pulmonis into pregnant rats results in high levels of bacterial infection in amniotic fluid, placenta and endometrium as well as histological placentitis. 14 Future studies comparing the pathophysiology of infection by the intravaginal route with the intravenous route may be valuable for separating clinical from subclinical aspects of intrauterine infection. This is particularly relevant as the clinical course of genital mycoplasmosis in humans is most closely linked to clinically silent chorioamnionitis rather than febrile intrauterine infections. 18 This study suggests that increased production of proinflammatory cytokines and MMP-9 occurs during genital mycoplasmosis but this is not sufficient for induction of PPROM, preterm labor or low birthweight in this model system. During spontaneous labor in monkeys, ProMMP-9 increases as labor approaches followed by increased production of activated MMP-9 (83 kDa). 8 It is also possible that MMP-9 must be transformed to its active form in order for it to digest membrane collagen and that the enzymes which activate proMMP-9 are not increased in this model.

Premature rupture of the membranes

Epidemiologic and molecular investigations of genital mycoplasmas from women and neonates at delivery

Possible role of Ureaplasma urealyticum in preterm premature rupture of the fetal membranes

Genital mycoplasmas stimulate tumor necrosis factoralpha and inducible nitric oxide synthase production from a murine macrophage cell line

Experimental primate model for Mycoplasma hominis chorioamnionitis and preterm labor

Experimental primate model for ureaplasma chorioamnionitis and preterm labor

III: 92-kd type IV collagenase (matrix metalloproteinase-9) activity in human amniochorion increases with labor

Identification of matrix metalloproteinase-9 in amniotic fluid and amniochorion in spontaneous labor and after experimental intrauterine infection or interleukin-1 beta infusion in pregnant rhesus monkeys

116-121 ª 2007 The Authors induced in rat amnion immediately prior to parturition

Production of matrix metalloproteinase-9 in lipopolysaccharide-stimulated human amnion occurs through an autocrine and paracrine proinflammatory cytokine-dependent system

Distinct molecular events suggest different pathways for preterm labor and premature rupture of membranes

Genital mycoplasmosis in rats: a model for intrauterine infection

Experimental genital mycoplasmosis causes increased levels of mRNA for IL-6 and TNF-alpha in the placenta

Placental lesions caused by experimental infection of Sprague-Dawley rats with Mycoplasma pulmonis

Prevention of pretmature birth

Microbial invasion of the amniotic cavity with Ureaplasma urealyticum is associated with a robust host response in fetal, amniotic, and maternal compartments

Role of tumor necrosis factor-alpha in the premature rupture of membranes and preterm labor pathways

Chorioamnionitis and colonization of the newborn infant with genital mycoplasmas