Mature female Sprague-Dawley rats (250 g; Harlan, Madison, WI) exhibiting regular 4-day to 5-day estrous cycles were housed in an environmentally controlled room with a 14L:10D cycle. All rats were allowed an acclimation period in the vivarium for 14 days (1 wk for acclimation and 1 wk to confirm reproductive cyclicity by evaluation of vaginal cytology) before any procedure was performed. The experiments were conducted with the approval of the University of Missouri Institutional Animal Care and Use Committee and in accord with the National Research Council's Guide for the Care and Use of Laboratory Animals (Washington, DC: National Academy Press; 1996).
Fluids in the Peritoneal Cavity Traverse the Reproductive Tract
First, we validated the hypothesis that peritoneal fluid, including secretions from endometriotic lesions, can enter, traverse, and influence the whole reproductive tract. Methylene blue, a dye commonly used to evaluate tubal patency and endometrial architecture in women, was used to track the course of fluids in the peritoneal cavity through the rat oviduct and uterus. One milliliter of a sterile nontoxic methylene blue solution (1% in water) was injected into the peritoneal cavity of rats with normal reproductive cycles (n = 15) using a sterile 1-ml syringe with a sterile 26-gauge needle. Rats were then euthanized by routine CO2 overdose at time points of 6, 12, 24, 48, and 72 h. The reproductive tracts were collected and evaluated for oviductal and uterine endometrial blue staining. At 6 h, the exterior surfaces of most abdominal organs, including the reproductive tract, were stained blue by the dye. This effect diminished as time increased and was absent at 48 h. Between 12 and 24 h, the blue dye could be seen migrating into the uterotubal junction, traversing about one third the distance of the uterine horn. Between 24 and 48 h, the dye had migrated about two thirds the distance of the horn, and at 72 h the dye could be seen exiting the cervices. Hence, the results of this study showed that peritoneal fluid does in fact bathe the ovary, enter the oviducts, and traverse the reproductive tract of rats ().
FIG. 1. A) Intact uterine horns, dye exiting cervices. B) One uterine horn opened to show dye present throughout horn. Peritoneal fluid traverses the oviduct and uterine horn. In 72 h, blue dye injected into the peritoneal cavity moved through the oviduct and (more ...)
Surgical Induction of Endometriosis
Endometriosis was surgically induced in Endo rats as previously described by Vernon and Wilson [27
] and as routinely performed in our laboratory [47
]. Animals were anesthetized with isoflurane (Butler Animal Health, St. Joseph, MO). Under aseptic conditions, the abdominal cavity was entered through a small 3- to 4-cm midventral incision initiated 2 cm above the urethral opening.
Briefly, unilateral hemihysterectomy and hemiovariectomy were performed. The right ovary and distal two thirds of the uterine horn were ligated with silk suture (American Cyanamid Co., Danbury, CT), surgically removed, and placed in warm (37°C) sterile PBS. The uterine tissue was trimmed of excess fat, bisected along its longitudinal axis, and cut into 2-mm squares. Four uterine squares (implants) were autotransplanted to the arterial cascades of the small intestine beginning at the cecum with 4–0 nonabsorbable nylon suture (Ethicon, Inc., Somerville, NJ). Sham-operated control rats (Shams) were subjected to unilateral hemihysterectomy and hemiovariectomy without the autotransplantation of uterine squares.
After completion of the surgical procedures, the abdominal cavity was rinsed with sterile PBS containing penicillin (100 IU/ml) and streptomycin (100 μg/ml) to hydrate the animal, lessen potential surgical adhesion formation, and help prevent infection. The muscle wall was closed with absorbable 3–0 suture (Ethicon, Inc.), and the skin was closed with 5-mm wound clips (MikRon; Becton Dickinson and Company, Sparks, MD). Rats received buprenorphine (Buprenex, 0.03 mg/kg; Rickitt & Coleman, Richmond, VA) subcutaneously for pain relief. One week after surgery, the wound clips were removed. Experimentation began after 4 wk, which were allotted for recuperation and development of endometriotic implants.
Four weeks after surgery, vaginal cytology was monitored daily in all rats as an index of reproductive cyclicity [52
]. The first group of cycling Endo and Sham rats was euthanized in a CO2
chamber between 700 and 800 h on the morning of estrus to collect unfertilized oocytes following LH surge between 1700 and 1900 h the prior evening. Additional Endo and Sham rats were cocaged with proven breeder males overnight on the evening of proestrus. Mating was confirmed the next morning by the presence of a vaginal plug or the presence of spermatozoa in a vaginal lavage. Pregnant rats were euthanized the next morning (Day 1) to collect zygotes, on Day 5 to collect preimplantation embryos/blastocysts, or on Day 15 of gestation to evaluate pregnancy loss or were allowed to gestate to term (pups used in experiment 5).
Experiment 1: Effects of Endometriotic Implants on Peritoneal TIMP1 Concentrations
The concentration of TIMP1 in the peritoneal fluid of nonpregnant estrus-stage rats (five Endo and six Sham) and of Gestational Day 1 pregnant rats (five Endo and four Sham) was quantified. Rats were euthanized, 1 ml of warm PBS was injected into the peritoneal cavity, and the abdomen was gently massaged for 30 sec. The peritoneal fluid washings were then aspirated using a 1-ml syringe, centrifuged for 5 min at 4°C to remove residual cells, and stored in a −80°C freezer.
Peritoneal fluid TIMP1 concentrations were quantified using an ELISA for rat TIMP1 per the manufacturer's instructions (RayBiotech, Inc., Norcross, GA) and were normalized on a total protein basis using the DC Protein Assay according to the manufacturer's instructions (BioRad Laboratories, Hercules, CA). Results were reported as the ratio of TIMP1 (in nanograms per milliliters) to total protein (in milligrams per milliliter). Differences in peritoneal fluid TIMP1 concentrations between Endo and Sham rats were analyzed using two-way ANOVA as follows: model TIMP1 concentration = condition (Endo or Sham) × pregnancy status (estrus or Day 1) × interaction. Tukey post hoc testing was used for pairwise multiple comparison procedures.
Experiment 2: Effects of Endometriotic TIMP1 on Ovarian Function
Ovarian function during the estrus stage and at Day 1 of pregnancy was evaluated in additional Endo (n = 11) or Sham (n = 13) rats (experiment 2a). The remaining right ovary was excised, weighed, and cut in half, and one half was placed into 10% neutral buffered formalin for 24 h. The fixed ovary was then rinsed in PBS, routinely paraffin embedded, and serial sectioned at 8-μm intervals, placing three serial sections per slide. The first slide was stained with hematoxylin-eosin (H&E), and the next four slides were left unstained to evaluate TIMP1 protein localization by fluorescent immunohistochemistry. This pattern of sectioning and staining was repeated for the entire one half of the ovary. The other half of the ovary was snap frozen and stored at −80°C for future gene studies.
Differences in the numbers of follicles and CL and in the presence of LUFS between Endo rats (three estrus, three Day 5, and three Day 15) and Sham rats (five estrus, five Day 5, and three Day 15) were evaluated histologically and quantified morphometrically as described herein. Luteinization was confirmed by an increase in the cytoplasmic:nuclear ratio. Antral follicles and LUFS were counted in one section on every H&E-stained slide, whereas CL were counted on every sixth H&E slide. These morphometric calculations were based on size so that no antral follicle, CL, or LUF was counted more than once in each ovary [29
]. All morphologic and morphometric analyses were performed by the same investigator blinded to the study group. Differences in the numbers of follicles and in the numbers of CL between Endo and Sham rats were analyzed using two-way ANOVA as follows: model follicular number, CL number, or LUFS = condition (Endo or Sham) × day (estrus, Day 5, or Day 15 of gestation) × interaction. Tukey post hoc testing was used for pairwise multiple comparison procedures.
Localization of TIMP1 protein in the ovaries of Day 1 pregnant Endo (n = 5) and Sham (n = 5) rats was quantified to detect potential TIMP1 involvement in ovarian dysfunction in endometriosis (experiment 2b). Briefly, tissues were deparaffinized with xylene and rehydrated through serial dilutions of ethanol from 100% to 70%. Nonspecific antibody binding was blocked by incubation of the tissues in 1% normal horse sera (Vector Laboratories, Burlingame, CA) in PBS (pH 7.4) for 20 min before incubation with the primary antibody. Polyclonal rabbit anti-rat TIMP1 (1:100, 5 ng/ml; Cell Applications, Inc., San Diego, CA) antibodies were diluted in PBS containing 0.1% bovine serum albumin (BSA; Sigma Chemical Company, St. Louis, MO) and 1% normal horse serum in PBS for 1 h. Tissues were then washed in PBS three times for 5 min.
Ovarian tissue sections were incubated for 30 min with secondary antibody anti-rabbit IgG conjugated with Alexa Fluor 488 (1 μg/ml in PBS; Invitrogen, Carlsbad, CA). Tissues were washed for 5 min with PBS before adding a coverslip with antifade mounting medium containing 4′,6′-diamidino-2-phenylindole (DAPI) (Vector Laboratories) to reduce photobleaching and to counterstain cell nuclei. To validate these results, additional ovarian sections were incubated with the same TIMP1 primary antibody as before and then incubated with a biotinylated secondary antibody anti-rabbit IgG (50 μl/10 ml in PBS with 0.1% BSA; Vector Laboratories) for 30 min, and tissues were again washed in PBS for 5 min. Tissues were then incubated with Texas Red-conjugated avidin D (15 μg/ml diluted in PBS; Vector Laboratories) for 30 min and mounted as already described. Immunostaining of both sets of tissues was examined using an Olympus inverted IX-71 microscope (Olympus, Melville, NY) equipped for fluorescence imaging.
Immunofluorescent localization of TIMP1 in the ovarian follicular theca and CL was quantified using computer-assisted image analysis (ImageJ; National Institute of Mental Health, Bethesda, MD). The morphometric parameter known as area fraction was used to quantify immunofluorescent intensity per area [54
]. The perimeters of antral follicles and CL from the Endo and Sham rats were traced using the freehand selection tool. The area fraction for follicular theca and CL fluorescence was defined as the total area traced. All data were reported as mean intensity per area. Both follicular and CL TIMP1 area fractions of Endo and Sham rats were normally distributed and were compared using Student t
Experiment 3: Effects of Endometriotic Implants on Oocyte Quality and Pre-Embryo Development
Oocyte quality and preimplantation embryo development were evaluated in oocytes on the morning of estrus (nine Endo and three Sham), Day 1 zygotes (10 Endo and four Sham), and Day 5 blastocysts (six Endo and three Sham) from additional Endo and Sham rats. The ovaries, oviducts, and uteri were excised en masse and placed in a 15-ml conical tube containing warm PBS for transportation (<5 min) to the microscopy laboratory.
The contents of each tube were emptied into a 35-mm culture dish. The cumulus masses containing oocytes or zygotes were located in the swollen ampulla of the oviduct using a Nikon SMZU stereoscope (Nikon Instruments Inc., Melville, NY) dissecting microscope. The ampulla was nicked with a 26-gauge needle so that the cumulus mass was extruded. The Day 5 preimplantation blastocysts were obtained by inserting a tuberculin syringe filled with PBS at 37°C into the oviduct and by flushing with the PBS through the uterine horn and out the cervix. Using a sterile Pasteur pipette, oocytes, zygotes, and blastocysts were transferred to a previously prepared dish of 37°C PBS.
To remove the cumuli oophori, the oocytes and embryos were repeatedly pipetted for 30 sec in a wash of serum-free Tyrode albumin lactate pyruvate (TALP)-Hepes (Hepes-buffered Tyrode-containing lactate, 0.2 mM pyruvate, and 3 mg/ml of BSA) with 0.5% w/v hyaluronidase (Sigma Chemical Company) and were then washed with TALP-Hepes. The zona pellucida was removed by a 5-min incubation in TALP-Hepes supplemented with 0.5% polyvinylpyrrolidone (molecular weight, 40
000; Sigma Chemical Company) and 0.5% pronase w/v (Sigma Chemical Company). Oocytes and embryos were washed in 37°C PBS. Formaldehyde (10%; Sigma Chemical Company) was added slowly to the PBS until reaching a final concentration of 2%. Embryos and oocytes were fixed at room temperature for 40 min. After fixation, embryos were washed and permeabilized with 0.1% Triton X-100 (Sigma Chemical Company) and then incubated with 1% normal goat serum to block nonspecific antibody binding before immunolabeling. Oocytes and embryos were pooled by gestational day (Day 1 or Day 5) within group (Endo or Sham).
The number, morphology, and quality of the oocytes and embryos were evaluated using epifluorescence microscopy combined with differential interference contrast (DIC) microscopy. Metaphase II oocytes collected from estrus rats at 6 h after ovulation were evaluated for quality by applying morphologic criteria. Day 1 zygotes were evaluated to determine the number of pronuclei and the presence of sperm flagellum in the ooplasm. A combination of DAPI staining to evaluate nuclei (Molecular Probes, Eugene, OR) and DIC microscopy to visualize ooplasm-incorporated sperm flagellum was applied to avoid false detection of parthenotes (one small peripherally located pronucleus, one polar body, and no flagellum in ooplasm) as fertilized ova (two centrally located pronuclei with incorporated flagellum). Pre-embryos collected on Day 5 were evaluated using the DNA stain DAPI combined with mouse monoclonal anti-tubulin antibody E7 (Developmental Studies Hybridoma Bank, Iowa City, IA) to evaluate chromosome alignment, spindle structure (mitotic blastomeres), and nuclear integrity.
Photomicrographs were taken with a Nikon Eclipse 800 microscope (Nikon Instruments Inc.) equipped with Cool Snap camera (Roper Scientific, Tucson, AZ) and MetaMorph software (Universal Imaging Corp., Downington, PA). Data were archived on CD-ROM compact disks, and color was merged using Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA). Differences in the number of oocytes and the number of zygotes between Endo and Sham rats were determined using Student t-test.
Experiment 4: Effect of Endometriotic Implants on Fecundity
The effects of endometriotic implants on fetal loss were evaluated on Gestational Day 15. Endo (n = 8) and Sham (n = 7) rats were euthanized in a CO2 chamber. Uteri were excised en masse and placed in a 100-mm Petri dish. The numbers of viable and nonviable fetal sacs were counted and photographed. Differences in the numbers of viable and nonviable fetuses (spontaneous fetal absorption/resorption sites [SAbs]) between Endo and Sham rats were determined using Student t-test and Mann-Whitney U rank sum test, respectively. The proportions of viable and SAb fetuses in Endo vs. Sham rats were evaluated using Chi-square test. Differences between the crown rump length of viable Endo and Sham rats were determined using Mann-Whitney U rank sum test. Peritoneal concentrations of TIMP1 were again measured by ELISA. Differences between peritoneal fluid TIMP1 concentrations of Endo with SAbs, Endo without SAbs, and Sham without SAbs were measured by one-way ANOVA and Tukey post hoc testing. Furthermore, peritoneal fluid concentrations of TIMP1 were correlated to the presence and number of SAbs using Spearman rank order correlation.
Experiment 5: Multigenerational Effects of Endometriosis on Oocyte Quality and Preimplantation Embryo Development
Possible inherited reproductive anomalies as a result of maternal exposure to endometriotic lesions were assessed. Female pups from experiment 1 (F1 generation) were allowed to sexually mature and were bred to a second group of proven breeder males. Oocytes, zygotes, and blastocysts were collected from the F1 daughters of Endo (n = 9) and Sham (n = 9) rats and were evaluated as described for their mothers, the F0 generation. Furthermore, F1 daughters of Endo (n = 3) and Sham (n = 3) mothers were allowed to sexually mature, were bred, and were allowed to gestate to Day 15. The numbers of implantation sites and SAbs were counted and photographed.
All statistical tests described for each experiment were performed using the Sigma Stat package (Systat Software, Inc., Point Richmond, CA). P < 0.05 was considered significant. The data were normally distributed and were reported as the mean ± SD except where noted.