Establishment, Validation, and Application of Multispectral Imaging Technology to Studies of Bovine Reproductive Tract Development.

Abstract

Development of the bovine uterus begins prenatally and is completed postnatally with formation of endometrial glands. Disruption of this process during neonatal life can reduce adult uterine capacity to support pregnancy. However, little is known about mechanisms regulating bovine endometrial development. With a goal of defining molecular and cellular events associated with bovine endometrial histogenesis and cytodifferentiation from birth (postnatal day 0 = PND 0), objectives here were to establish methods for immunohistochemical (IHC) localization of progesterone receptor (PR-A, -B forms) and estrogen receptor (ESR1), as well as two markers of cell proliferation, Ki-67 and bromodeoxyuridine (BrdU), in developing bovine endometrium using multispectral imaging (MSI). For purposes of technical validation, uterine tissue was obtained from a single Holstein heifer treated with BrdU (5mg/kg BW/day, i.v.) for five days prior to euthanasia on PND 40. Tissue was fixed in 4% paraformaldehyde, embedded in Paraplast-plus, sectioned (6μm) and subjected to antigen retrieval in citrate buffer (pH 6). An indirect, non-amplified, multi-label IHC method was employed using primary antibodies chosen based upon target specificity, host species and immunoglobulin subtype. Matched, AlexaFluor-labeled secondary antibodies (Invitrogen Corporation; Carlsbad, CA) were used to produce target-specific fluorescent signals. All tissue sections were counterstained with POPO-1 to reveal nuclear DNA. Fluorescent staining for cytokeratin 8 was used to distinguish epithelium from stroma in image analyses. The Nuance FX MSI system (Caliper Life Sciences; Hopkinton, MA) was used to capture quantifiable image data at specific wavelengths from as many as four unique fluorescent signals simultaneously. Fluorescent signals were analyzed using CellProfiler (www.cellprofiler.com). Spectrally unmixed target signal intensities were compared between stromal and epithelial cell compartments on a within tissue basis after extraction of compartment-specific regions of interest from single- and multi-labeled tissue sections. Data were subjected to analysis of variance to evaluate technical sources of variation. Images generated using multi-label IHC and MSI procedures were equivalent, qualitatively and quantitatively, to those obtained with single labeling procedures. Using MSI, quantifiable, wavelength-specific data were extracted from spectrally unmixed images generated from multi-labeled tissues. Automated analysis procedures permitted rapid generation of unique and colocalized fluorescent signal intensities and/or labeling indices for ESR1, PR, Ki-67 and BrDU in stromal and epithelial cell compartments. Relative signal intensity for ESR1 and PR was higher (P<0.001) in epithelium than stroma. However, labeling indices for these targets were greater (P<0.0001) in stroma than in epithelium. Labeling idices for both cell proliferation markers, Ki-67 and BrdU, were greater (P<0.001) for epithelium than for stroma. Establishment and validation of multi-label IHC and MSI procedures for qualitative and quantitative analysis of multiple fluorescent signals in single tissue sections sets the stage for studies designed to evaluate patterns of neonatal bovine endometrial development at cellular and molecular levels simultaneously. [Support: AU-CVM AHDR and NSF-EPS 0814103.]