Myometrium

The Iso-Seq method for sequencing full-length transcripts was developed by PacBio during the same time period as the genome assembly. We therefore used this technique to improve characterization of transcript isoforms expressed in cattle tissues using a diverse set of tissues collected from L1 Dominette 0 1449 upon euthanasia. The data were collected using an early version of the Iso-Seq library protocol [26 ] as suggested by PacBio. Briefly, RNA was extracted from each tissue using Trizol reagent as directed (Thermo Fisher). Then 2 μg of RNA were selected for PolyA tails and converted into complementary DNA (cDNA) using the SMARTer PCR cDNA Synthesis Kit (Clontech). The cDNA was amplified in bulk with 12–14 rounds of PCR in 8 separate reactions, then pooled and size-selected into 1–2, 2–3, and 3–6 kb fractions using the BluePippin instrument (Sage Science). Each size fraction was separately re-amplified in 8 additional reactions of 11 PCR cycles. The products for each size fraction amplification were pooled and purified using AMPure PB beads (Pacific Biosciences) as directed, and converted to SMRTbell libraries using the Template Prep Kit v1.0 (PacBio) as directed. Iso-Seq was conducted for 22 tissues including abomasum, aorta, atrium, cerebral cortex, duodenum, hypothalamus, jejunum, liver, longissimus dorsi muscle, lung, lymph node, mammary gland, medulla oblongata, omasum, reticulum, rumen, subcutaneous fat, temporal cortex, thalamus, uterine myometrium, and ventricle from the reference cow, as well as the testis of her sire. The size fractions were sequenced in either 4 (for the smaller 2 fractions) or 5 (for the largest fraction) SMRTcells on the RS II instrument. Isoforms were identified using the Cupcake ToFU pipeline [27 ] without using a reference genome.
Short-read–based RNA-seq data derived from tissues of Dominette were available in the GenBank database because her tissues have been a freely distributed resource for the research community. To complement and extend these data and to ensure that the tissues used for Iso-Seq were also represented by RNA-seq data for quantitative analysis and confirmation of isoforms observed in Iso-Seq, we generated additional data, avoiding overlap with existing public data. Specifically, the TruSeq stranded mRNA LT kit (Illumina, Inc.) was used as directed to create RNA-seq libraries, which were sequenced to ≥30 million reads for each tissue sample. The Dominette tissues that were sequenced in this study include abomasum, anterior pituitary, aorta, atrium, bone marrow, cerebellum, duodenum, frontal cortex, hypothalamus, KPH fat (internal organ fat taken from the covering on the kidney capsule), lung, lymph node, mammary gland (lactating), medulla oblongata, nasal mucosa, omasum, reticulum, rumen, subcutaneous fat, temporal cortex, thalamus, uterine myometrium, and ventricle. RNA-seq libraries were also sequenced from the testis of her sire. All public datasets, and the newly sequenced RNA-seq and Iso-Seq datasets, were used to annotate the assembly, to improve the representation of low-abundance and tissue-specific transcripts, and to properly annotate potential tissue-specific isoforms of each gene.

The uterine junctional zone was defined as the inner third of the myometrium [24 (link)]. All samples containing endometrium and junctional zone were collected from the mid-fundal and isthmic areas of the uterine anterior wall. The uterus was opened along the sagittal plane after surgery. Multiple 1 × 1 × 1 cm³ samples were collected and fixed in buffered formaldehyde and processed routinely for paraffin embedding. Serial 4 μm sections were prepared from each paraffin-embedded tissue block and handled for immunohistochemical staining. All uterine samples were examined by optical microscope to confirm presence of endometriosis, absence of adenomyosis and respective phases of the menstrual cycle. Under low light microscopy magnification, the uterine junctional zone was located just 3 mm below the endometrium [20 (link)].
After routine de-paraffinization and rehydration procedures, the slides were heated in a microwave oven (700 W) in citrate buffer saline (9.0) for twelve min and cooled at room temperature for antigen retrieval. Every sections were incubated with a drop of 3% H₂O₂ deionized water (PV-6000, Wuxi, China) for 20 min at 37 °C temperature. After 2 washes with phosphate-buffered saline (PBS), the slides were hatched with polyclonal rabbit anti-OTR (1:100 dilution, bs-1314R, Bioss, Beijing, China) overnight at 4 °C refrigerator. After 3 washes with phosphate-buffered saline (PBS), the sections were incubated with biotinylated anti-rabbit immunoglobulin G (1:400) for 30 min at room temperature. The bound antibody complexes were stained for 3 mins with diaminobenzidine. The slides were then washed, counterstained with hematoxylin, dried and mounted. Negative control sections were processed by omitting the primary antibody. Myometrium of pregnant uterus were used as positive controls. Immunoreactivity staining was characterized quantitatively by digital image analysis on the Image Pro-Plus 6.0 (Nikon, Japan). Images were obtained with a microscope fitted with a digital camera. A series of 4 random images on several sections were taken for each immunostained parameter to obtain a mean value. Staining was defined by color intensity, and a color mask was made. The mask was then applied equally to all images, and measurements were obtained. Immunohistochemical parameters were assessed in the area detected by total optical density and mean optical density, which is equivalent to the intensity of staining in the positive cells.

Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118–125 days gestation (term = 172 days) received one of two experimental treatments: 1) choriodecidual and intra-amniotic saline infusions (n = 5), or 2) GBS choriodecidual inoculation (n = 5). Saline infusions were performed separately in the choriodecidual and amniotic fluid to confirm that saline would not stimulate cytokine production in either compartment. In two saline controls, fetal samples were not collected due to either an inability to place the fetal catheter during initial surgery or clotting of the fetal catheter. This resulted in three fetal cytokine analyses in the saline group. In one GBS case, technical problems led to only intermittent data collection and so the remaining uterine activity data was excluded for this animal.
In our model, pregnant pigtail macaques were time-mated and fetal age determined using early ultrasound. The tethered chronic catheter preparation was used for all in vivo experiments and is a major breakthrough in studying maternal-fetal immunologic responses [53] (link), [54] (link). The animal was first conditioned to a nylon jacket/tether system for several weeks before surgery, which allowed free movement within the cage, but protected the catheters. On day 118–125 of pregnancy (term = 172 days) catheters were implanted into the maternal femoral artery and vein, fetal internal jugular vein, amniotic cavity, and choriodecidual interface in the lower uterine segment (between uterine muscle and fetal membranes, external to amniotic fluid). Fetal ECG electrodes and a maternal temperature probe were also implanted. After surgery, the catheters/electrodes were tracked through the tether system and cefazolin and terbutaline sulfate were administered to reduce postoperative infection risk and uterine activity. Both cefazolin and terbutaline were stopped at least 72 hours before experimental start (∼5 half-lives for terbutaline, 40 half-lives for cefazolin, >97% of both drugs eliminated). Cefazolin 1 gram was administered intravenously each day in saline controls to minimize chances of a catheter-related infection. Experiments began approximately two weeks after catheterization surgery to allow recovery (∼30–31 weeks human gestation). At our center, term gestation in the non-instrumented pigtail macaque population averages 172 days.

The method for the measurement of uterine contraction has been previously described (15 (link)). Briefly, the uterine muscle strips are fixed to the constant temperature bath and multi-channel physiological signal acquisition and processing system while continuously ventilated with 95% O₂ and 5% CO₂ at 37.0 ± 0.5°C. Our previous study has confirmed that uterus strip contractility performance is most appropriate at a 2-g stretch (15 (link)). After the appearance of a stable and regular contraction curve, the strips were stimulated with 96 mM KCl and different concentrations of oxytocin (from 10^-11 to 10^-6 mM), and the contraction response was recorded. Finally, the uterine muscle strips were weighed at the end of the experiment for calibration.
Quantitative analysis of the contractility of the uterine muscle strips was performed by a multichannel physiological signal acquisition system (RM6240E, Chengdu, China) by calculating the area under the curve (AUC) of the contraction curve presented as AUC/g tissue weight. The AUC was measured at time 0 and was subtracted from the AUC measured after 5 min of application of KCl or each oxytocin concentration.

Isolation of primary human gestational uterine smooth muscle cells was achieved by the enzymatic dispersion method. Myometrium was obtained from women after elective cesarean delivery in late pregnancy. The endometrium and epithelium were slightly scraped off the surface of the myometrium with a sterile blade, and the myometrium was then cut up with tissue scissors. The cut uterine tissue was digested using 15 ml of digestion solution, followed by shaking for 1 h at 37°C on a shaker. Then, the digested solution was filtered through a 100-μm filter to remove the tissue fragments, and the filtrate was transferred to a sterile centrifuge tube and centrifuged at 1000 rpm/min for 5 min before discarding the upper layer. The cell precipitate was resuspended in normal glucose medium, and the cell suspension was placed in a 25 cm² cell culture flask (Corning) and incubated at 37°C, 5% CO₂ incubator until fusion.

Total protein was extracted with RIPA lysis buffer containing benzoyl fluoride and phosphatase inhibitors (Beyotime Biotechnology, China) from uterine muscle strips. The supernatant was collected by centrifugation at 4°C and 12,000 rpm/min for 10 min. The BCA protein assay was used to determine the total protein concentration. The supernatant and loading buffer were mixed (1:4) and heated at 100°C for 10 min. Protein homogenates were electrophoresed on 10% SDS (sodium dodecyl sulfate) polyacrylamide gels and then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were incubated in PBS-Tween buffer containing 5% skim milk for 2 h to block non-specific sites and then incubated overnight at 4°C in primary antibody solution containing the monoclonal mouse antibody to GAPDH (1:5000, Abcam, England) or the monoclonal rabbit antibody to TREK1 (1:200, Sigma-Aldrich, American) or HIF-1α (1:500, Abcam, England). The PVDF membranes were then washed 3 times in PBS-Tween for 15 min each and then incubated with horseradish peroxidase-coupled goat anti-rabbit secondary antibody (1:10,000, Abcam, England) or goat anti-mouse secondary antibody (1:10,000, Abcam, England) for 2 h. The membrane blots were washed with PBS-Tween and visualized by enhanced chemiluminescence (ECL, Biosharp, China). A band of 37 kDa for GAPDH, 110 kDa for HIF-1α, and 47 kDa for TREK1 was detected according to the corresponding protocols of the antibody products and were confirmed by the Marker. GAPDH was used as the internal control. Reaction bands corresponding to GAPDH, TREK1, and HIF-1α were analyzed with Image J.