Three experiments were performed (). 1) TIMP1 was modulated in vitro to determine its effect on embryo development. 2) TIMP1 was modulated in vivo in Endo and Sham rats to determine the effect on embryo development and ovulation. 3) Control rats were treated with peritoneal fluid from Endo and Sham rats to determine the specificity of TIMP1 effects on ovulation.
FIG. 1. In vitro and in vivo effects of modulating TIMP1 on preimplantation embryo development and ovulation. In experiment 1, zygotes were collected from control rats and cultured for 24 h in the presence and absence of TIMP1. In experiment 2, Endo rats were (more ...)
All the experiments were conducted with the approval of the University of Missouri Institutional Animal Care and Use Committee and in accordance with the National Research Council's “Guide for Care and Use of Laboratory Animals.” Mature female Sprague-Dawley rats (250 g; Harlan Laboratories, Inc., Madison, WI) exhibiting normal 4–5 d estrous cycles were housed in an environmentally controlled room with a 14L:10D cycle. Rat chow and water were available ad libitum. Rats were acclimated to the vivarium for 14 d prior to surgery.
Experiment 1: Modulation of TIMP1 During In Vitro Embryo Culture
To first determine if TIMP1 concentrations found in the PF of rats with endometriosis were detrimental to preimplantation embryo development, a pilot study was performed. Day 1 zygotes were collected from normal rats (no surgery) and cultured in vitro ().
To stimulate folliculogenesis and follicular maturation, immature Sprague-Dawley rats (n = 4, 50–60 g; Harlan Laboratories, Inc.) were injected with PG 600 (20 international units [IU]; Intervet, Millsboro, DE) and 2 days later injected with hCG (40 IU; EMD Biosciences, Gibbstown, NJ). The females were then cocaged with proven breeder males.
Females were euthanized the next afternoon in a CO2
chamber followed by a cervical dislocation. The ovaries and oviducts were excised and rinsed twice in HEPES-buffered Tyrode solution as described previously [36
]. The oviducts were removed and the ampulla was nicked, allowing the release of the cumulus-oocyte complexes. Cumulus cells were removed using hyaluronidase in HEPES buffer (1 mg/ml; Sigma Aldrich, St. Louis, MO).
Embryos were cultured for 24 h to the two-cell stage in rat embryo culture media (protein-free minimal essential medium with amino acids, a kind gift of Dr. John Critser, University of Missouri) with (n = 16 embryos) or without (n = 6 embryos) recombinant rat TIMP1 (700 ng/ml; R&D Systems, Minneapolis, MN). This concentration of TIMP1 protein in the culture media is equivalent to PF TIMP1 concentrations in the endometrium of rats, which has been previously correlated with ovarian anomalies and subfertility [17
After culture, removal of the zona pellucida, fixation and processing for immunolabeling was performed as described previously [17
]. Two-cell embryos were evaluated for TIMP1 and nuclear pore complex protein localization. A mouse monoclonal antinuclear pore complex antibody MAb 414 (Abcam, Cambridge, MA) was used to evaluate nuclear envelope reassembly reflective of the blastomere/embryo quality. A rabbit polyclonal anti-TIMP1 antibody (Cell Applications, Inc., San Diego, CA) was used to detect TIMP1 in the embryos while nuclear localization was visualized by the DNA stain 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA). Species-appropriate, fluorescently conjugated secondary antibodies or biotin-conjugated antibodies followed by avidin-conjugated fluorescent labels were then used to detect primary antibodies. TIMP1 and nuclear pore complex localization were visualized by epifluorescence microscopy.
Photomicrographs were taken with a Nikon Eclipse 800 microscope (Nikon Instruments Inc., Melville, NY) equipped with Cool Snap camera (Roper Scientific, Tucson, AZ) and MetaMorph software (Universal Imaging Corp., Downington, PA), and color merged using Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA). In addition to immunofluorescence, embryo quality was assessed by gross morphological analysis. Embryos were considered of good quality if they had two equal-sized blastomeres free of cytoplasmic fragmentation or granularity, normal blastomere adhesion, and normal appearing nuclei. Embryos were considered abnormal if any of these qualifications were not met.
Induction of Endometriosis
For experiments 2 and 3, endometriosis was surgically induced in rats (Endo) as previously described by Vernon and Wilson [13
] and routinely performed in our laboratory [19
]. A unilateral ovariohysterectomy was performed, and sections of the removed uterus were autotransplanted into the arterial cascade of the small intestine. The remaining rats underwent a control surgery (Sham). Sham rats also had a unilateral ovariohysterectomy without autotransplantation of the uterine squares. After 4 wk of endometriotic lesion growth, reproductive cycles were synchronized but not superovulated with a LHRH agonist (40 μg/250 g rat; Sigma) to allow treatments to be administered on the same day. Reproductive cyclicity was monitored by evaluation of vaginal cytology. Only rats in estrus were used in this study.
Experiment 2: In Vivo Modulation of TIMP1
Prior studies from our laboratory [17
] have demonstrated methylene blue dye injected into the peritoneal cavity can bathe the ovary, enter the oviducts, and traverse the reproductive tract. Therefore, experiments 2 and 3 used an intraperitoneal route of treatment delivery.
Sham rats (n = 5 per treatment) received intraperitoneal injections of recombinant rat TIMP1 (2.8 μg/kg; R&D Systems), GM6001 (N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide), a synthetic broad-spectrum MMP inhibitor (3.9 mg/kg; Millipore; Billerica, MA), or PBS as a control. The recombinant TIMP1 dose was determined from concentrations of intraperitoneal TIMP1 previously reported in Endo rats [17
]. The GM6001 dose was found to be efficacious at inhibiting many MMPs reported in an in vivo study of nude mice [39
] and was then adjusted for rat body weight.
Endo rats (n = 5 per treatment) received intraperitoneal injections of a function-blocking TIMP1 antibody, (32 μg/kg rat; AbD Serotec; Raleigh, NC) or vehicle. The dose of TIMP1-blocking antibody originated from an in vivo study in male mice adjusted for rat body weight and was validated as the most efficacious dose to reduce TIMP1 in PF of control rats in our laboratory [40
Treatments (intraperitoneal injections) started on the morning of the second proestrus after synchronization and were repeated every third day for a total of three injections. Rats were then caged with proven breeder males on the subsequent evening of proestrus. Mating was confirmed the next morning by the presence of a vaginal plug or the presence of sperm in a vaginal lavage, and this was considered as Day 1 of pregnancy. To determine differences in quality and development, preimplantation embryos were collected from each rat and fixed as described.
Evaluation of Embryo Quality Following In Vivo Modulation of TIMP1
Zygotes were evaluated by using the DNA stain DAPI combined with mouse monoclonal antitubulin antibody E7 (Developmental Studies Hybridoma Bank, Iowa City, IA) to evaluate chromosome alignment, spindle structure (mitotic blastomeres), and nuclear integrity as well as with a rabbit polyclonal antiproteasome antibody to detected the distribution and abundance of proteasomes, which is reflective of embryo quality. Epifluorescence microscopy combined with differential interference contrast (DIC) microscopy was used to analyze the zygotes. Proteasome intensity was quantified morphometrically using Image J (NIH, Bethesda, MD) by tracing each zygote and measuring pixel intensity of the proteasome channel after the background was subtracted.
Determination of the Effect of In Vivo Modulation of TIMP1 in Follicle and CL Numbers
Differences in the numbers of follicles and CLs between treatment groups were evaluated histologically and quantified morphometrically as described previously [15
]. These morphometric calculations were based on size so that no antral follicle, CL, or luteinized unruptured follicle (LUF) was counted more than once in each ovary [15
]. All the morphological and morphometric analyses were performed by two investigators blinded to the study group. Differences in the numbers of follicles and in the number of CLs between treatments were analyzed by one-way ANOVA and Tukeys test for posthoc pairwise multiple comparisons.
Immunolocalization of TIMP1 in the Ovaries
Localization of TIMP1 protein in the ovaries was quantified to detect potential TIMP1 involvement in ovarian dysfunction in endometriosis. Immunohistochemistry was performed as previously described [17
] except that the tissues were blocked in 1% hydrogen peroxide in methanol for 1 h. Slides were incubated with polyclonal rabbit anti-rat TIMP1 (1:100, 5 ng/ml, Cell Applications, Inc.) for 1 h and then with secondary antibody, an anti-rabbit IgG conjugated with Alexa Fluor 488 (1 μg /ml in PBS; Invitrogen) for 30 min. DAPI (Vector Laboratories, Burlingame, CA) was used to counterstain cell nuclei.
Immunofluorescent localization of TIMP1 in the ovarian follicular theca and CL was quantified morphometrically by area fraction as described previously [17
]. All the data were reported as mean intensity per area. Both follicular and CL TIMP1 area fractions of treatment groups were normally distributed and compared using one-way ANOVA and Tukey posthoc pairwise multiple comparison procedures.
TIMP1 Protein Inhibitory Activity
To assess the inhibitory activity of TIMP1, reverse zymography was performed using a kit and protocol established by Dr. Dylan Edwards (University of East Anglia, Norwich, U.K.). To protect the integrity of the extracellular matrix degrading enzymes, protein extraction was performed using detergents instead of heat as described by Hu et al. [41
]. Supernatants were then collected, and an aliquot was subjected to the DC Protein Assay (Bio-Rad Laboratories, Richmond, CA). Samples were either used immediately or stored at −80°C until use.
Protein samples (5 μg) were separated by SDS-PAGE using 12% acrylamide with gelatin (0.1% w/v) gels and gelatinases A and B (MMP2 and 9, respectively). A TIMP1 standard was included on each gel as a positive control. As the gel was destained, areas in the gel where TIMPs inhibited gelatinolytic activity were visualized as dark blue bands while the remainder of the gel lightened due to the enzyme's ability to break down the gelatin in the gel. This method was used to visualize free TIMPs but not MMP-TIMP complexes in the ovarian extracts.
Western blot analyses were performed to confirm the results of reverse zymography as previously described [42
]. Total protein (5 μg) was separated by a 12% SDS gel and transferred to a nitrocellulose membrane. The membrane was incubated with polyclonal rabbit anti-rat TIMP1 (1:100, 5 ng/ml; Cell Applications, Inc.) overnight. A biotinylated secondary antibody, anti-rabbit IgG (50 μl/10 ml), was used before visualization of the bands by an ABC kit (Vector Laboratories) with diaminobenzidine as per the manufacturer's instructions.
Quantification of Ovarian Timp1 Gene Transcript Levels
Timp1 mRNA levels in the ovaries were quantified by performing quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA was isolated from one-quarter of the ovary (~25 mg) using the Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA) as per the manufacturer's instructions, including the optional on-column DNase digestion step with RNase-free DNase. The quantity and quality of the RNA were checked using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA).
Ovarian RNA (500 ng) was reverse transcribed into cDNA using the First Strand cDNA Synthesis Kit for qRT-PCR (Roche, Basel, Switzerland) as per the manufacturer's instructions. The cDNA was diluted 10-fold prior to quantification by qRT-PCR.
Quantitative RT-PCR was performed using Taqman PCR Mix from Applied Biosystems (Foster City, CA) in an ABI 7500 Real-Time PCR System (Applied Biosystems); 18s
gene expression was used as the endogenous control to normalize the target gene expression data. Primer/probe sets for Timp1
were purchased from Applied Biosystems. Normal rat estrus-stage uterus cDNA served as the arbitrary constant. Relative mRNA expression for Timp1
was calculated as described previously [43
]. One-way ANOVA was used to detect statistically significant differences.
Ovarian Timp1 Transcript Localization
To localize the source of the Timp1 mRNA in the ovary, in situ hybridization was performed. All in situ hybridization was performed on paraffin-embedded tissues that were processed and sectioned specifically for the in situ hybridization. Slides were deparaffinized, rehydrated, and transferred to a pretreatment buffer of 10 mM Tris, 1 mM ethylenediaminetetraacetic acid, and 0.05% tween 20 (polysorbate 20, pH 7.6; Fisher Scientific, Pittsburgh, PA) at 95°C for 20 min to permeabilize the tissues for hybridization. Peroxidases were blocked before hybridization of the probe. Oligonucleotide probes were designed for a sense (AGC CCT TAT AAC CAG GTC CG) and antisense (CGG ACC TGG TTA TAA GGG CT) DNA probe with 100% identity to the exon spanning rat Timp1. Tissues were hybridized overnight at 37°C with a hybridization buffer containing 20% 20× SSC (3 M NaCl, 300 mM trisodium citrate, pH 7.0), 10 mg/ml bovine serum albumin, 20% (v/v) dextran sulfate (Fisher Scientific), 10 mg/ml sonicated salmon sperm (Invitrogen), and the oligonucleotide probe at 200 ng/ml. The remainder of the procedure was followed using the Vector Laboratories FISH protocol.
Experiment 3 In Vivo Treatments with PF
To determine the specificity of peritoneal endometriotic TIMP1 to the mechanisms of endometriosis-associated infertility, peritoneal washings from the vehicle-treated Endo and Sham rats were collected from the in vivo experiments (). Endo rat PF was pooled (n = 5) and divided. TIMP1 was immunoprecipitated from one-half with a mouse monoclonal anti-rat TIMP1(R&D Systems) attached to immobilized protein A-Trisacryl beads (Fisher Scientific) as per the manufacturer's instructions. Sham rat PF was also pooled (n = 5).
TIMP1 concentration in each pooled PF was tested by ELISA (RayBiotech, Inc., Norcross, GA) and for total protein as described above. About 10.8 mg of total protein/ml from each pooled peritoneal fluid group was snap frozen and lyophilized overnight. Lyophilized PF was reconstituted in sterile water for injection to normalize the amount of protein and amount of treatment injected into each treated rat.
Additional rats without surgery (n = 6 per treatment) were reproductively synchronized as before and injected with PF treatments after their second proestrus. Rats were euthanized the next morning (morning of estrus). Ovaries from these rats were collected and prepared as described before.
Differences in the numbers of follicles and CLs between treatment groups were evaluated histologically and quantified morphometrically. Localization of TIMP1 protein in the ovaries was detected and compared using one-way ANOVA and Tukey posthoc pairwise multiple comparison procedures. Also, gene transcript levels of Timp1 were determined in these ovaries to compare the specificity of endometriotic peritoneal TIMP1′s role in TIMP1 expression in the ovary; the levels were analyzed using a Kruskal-Wallis ANOVA on ranks.
All the statistical tests described for each experiment were performed using the Sigma Stat package (Systat Software, Inc., Point Richmond, CA). A P value of < 0.05 was considered significant. The data were normally distributed and were reported as the mean ± the standard deviation (SD) except where noted.