Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition, non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells.

Transcript

Different oviduct segments have distinct physiological functions and are differentially susceptible to pathological change. Identification of the different segments in a very small mouse oviduct is challenging, as is the production of a good yield of well-differentiated cells by tissue dissociation. In this video, we will demonstrate how to uncoil the mouse oviduct, recognize the different segments so that they can be analyzed individually, and finally, how to produce a relatively high yield of differentiated dissociated cells.

A better understanding of the environment specific to the ampulla may improve in vitro fertilization techniques. Also, by contrasting the infundibulum with other segments, we may better understand the infundibulum's propensity for malignant transformation. The single-cell dissociation procedure liberates stromal, as well as epithelial cells from the oviduct.

Our method also provides opportunities to separately analyze immune, smooth muscle, and epithelial cell contributions to physiology and pathology of the oviduct. Working under a dissection microscope can be difficult at first. I would advise practicing with a larger tube such as the uterus to ensure that your tools are adequate for the oviduct microdissection.

Begin by affixing a piece of dental wax to a Petri dish with adhesive and letting it dry. Then sanitize the dish with 70%ethanol. The surface is now ready for dissection.

Immobilize the Petri dish containing the dental wax on the cold platform under a dissection microscope when ready for dissection. Disinfect the ventral surface of the animal with 70%ethanol. Then open the abdominal and pelvic cavity with sterile dissection scissors.

Move the gastrointestinal tract to one side to locate the dorsal bihornal uterus. Follow each uterine horn rostrally to locate each oviduct and ovary that are enveloped in the uterine fat pad just below the kidney. Then excise both lateral oviducts with the ovaries and a portion of the distal uterine horn still attached using dissection scissors.

Cut each lateral uterine horn with approximately one to two centimeters of the horn still attached to the coiled oviduct and ovary. Submerge the tissue immediately in two milliliters of cold dissection medium, then affix the uterine tissue to the dental wax with a sterile 25 gauge needle to secure tissue for dissection. Clean and dissect the ovarian fat pad and connective tissue to visualize the oviduct clearly.

Use a one milliliter syringe to dispense one to two drops of sterile 1%toluidine blue dye solution and incubate for 30 seconds to one minute. Rinse with cold DPBS and then remove all the liquid. Lightly pull the ovary from the coiled oviduct.

Cut at the bursal membrane in the broad ligament to remove the ovary from the oviduct without compromising the tubal tissue. Locate the distal fimbrial end of the oviduct found lateral to the uterine tubal junction. Gently pull the tube and cut away at the mesosalpinx with springform micro-scissors to uncoil the oviduct.

Cut the tube to produce the infundibular region. Then excise the ampullary region by cutting between turns two and three. Finally, cut the remaining portion to the uterine tubal junction which is the isthmic region.

Immediately snap freeze the dissected tissue segments in liquid nitrogen and then add 200 microliters of cold RNA extraction reagent and one-to-one homogenization bead mix to the tissue for RNA isolation. Starting at the fimbrial region, slit the tube longitudinally with springform scissors and use forceps as leverage to expose the luminal epithelium. Mince the slit open portions and the remaining oviduct into approximately one to two millimeter segments.

Then add five milliliters of warm non-enzymatic dissociation buffer to the tissue and incubate at 37 degrees Celsius. Fluorescent and phase contrast images of a cell cluster positive for occludin, nuclei, and acetylated tubulin are shown here. Red denotes occludin, blue denotes nuclei, and green denotes acetylated tubulin.

The ciliated apical border remains intact throughout the protocol and withstands further digestion to single cells. In these representative images, merged, red, blue, phase contrast, and green channels are shown. After a brief pronase incubation, the majority of cells are single.

Propidium iodide staining shows approximately 93%viability in Brightfield, red channel PI positive, and merge. Inset on merge shows an intact ciliated border on a single dissociated cell. Such cells have actively beating cilia.

Whole RNA was assessed for yield and quality by microvolume spectrophotometer and bioanalyzer. Results show that this method yields 800 to 1, 200 nanogram RNA per segment except for infundibular samples where pooling from two animals may be necessary for appropriate yield for downstream assays. The presented results for infundibulum are pooled from two animals, totaling four infundibular regions.

This protocol successfully achieves pure RNA analyzed with a microvolume spectrophotometer and a bioanalyzer. Samples have a 260 by 280 nanometer absorption ratio between 1.8 and 2, typical of pure RNA nucleotide species. Bioanalyzing the samples showed high RNA integrity with assessed RNA integrity number above seven, appropriate for downstream expression and sequencing analysis.

Remember to retain one to two centimeters of the uterine horn to affix the tubal system to the dissection platform. In addition, the use of the toluidine blue dye helps in the fast uncoiling of the oviduct. The faster the uncoiling, the better the preservation and the least amount of RNA degradation.

This method is sufficient to obtain RNA of appropriate quality for RNA sequencing allowing individual segment sequencing. The cell dissociation is appropriate for single-cell sequencing or flow cytometry allowing a more precise segment and cell analysis. The method will help move the field beyond whole tube analysis that masks differences at the segment or single cell level.

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Journal (Developmental Biology)

Journal (Developmental Biology)

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

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