Volumetric imaging of the developing prepubertal mouse uterine epithelium using light sheet microscopy

Abstract

Endometrial or uterine glands secrete substances essential for uterine receptivity to the embryo, implantation, conceptus survival, and growth. Adenogenesis is the process of gland formation within the stroma of the uterus. In the mouse, uterine gland formation initiates at postnatal day (P) 5. Uterine gland morphology is poorly understood because it is primarily based on two-dimensional (2D) histology. To more fully describe uterine gland morphogenesis, we generated three-dimensional (3D) models of postnatal uterine glands from P0 to P21, based on volumetric imaging using light sheet microscopy. At birth (P0), there were no glands. At P8, we found bud- and teardrop-shaped epithelial invaginations. By P11, the forming glands were elongated epithelial tubes. By P21, the elongated tubes had a sinuous morphology. These morphologies are homogeneously distributed along the anterior-posterior axis of the uterus. To facilitate uterine gland analyses, we propose a novel 3D staging system of uterine gland morphology during development in the prepubertal mouse. We define five uterine gland stages: Stage 1: bud; Stage 2: teardrop; Stage 3: elongated; Stage 4: sinuous; and Stage 5: primary branches. This staging system provides a standardized key to assess and quantify prepubertal uterine gland morphology that can be used for studies of uterine gland development and pathology. In addition, our studies suggest that gland formation initiation occurs during P8 and P11. However, between P11 and P21 gland formation initiation stops and all glands elongate and become sinuous. We also found that the mesometrial epithelium develops a unique morphology we term the uterine rail.

Keywords: adenogenesis; epithelium; postnatal development; reproductive tract; uterus.

FIGURE 1

3D image analysis of the postnatal mouse uterus. Fluorescent light sheet images and 3D reconstructions of the postnatal mouse uterus and individual uterine glands. (a, e, i, m, q) TROMA-1 stained immunofluorescent images of the uterine epithelium. Mesometrial view. (b, f, j, n, r) Pseudo-colored surface renderings of the uteri corresponding to the first column. Mesometrial view. (c, g, k, o, s, u) Pseudocolored surface renderings of single uterine glands. (d, h, l, p, t, v) Optical cross-sections of fluorescent light sheet images of TROMA-1 stained uteri. White arrowheads correspond to the glands in the third column. Red arrowheads point to constricted region of teardrop-shaped gland. Anti-mesometrial to the right. (a–d) P0, (e–l) P8, (m–p) P11, and (q–v) P21. Scale bars: 100 μm

FIGURE 2

Uterine gland volume during postnatal development. Glands were measured with Imaris FilamentsTracer to quantify the volume of various gland types (bud, teardrop, elongated or sinuous) at P8, P11, and P21. Each dot, square, or triangle represents the volume of an individual gland. Error bars indicate average volume ±1 standard deviation

FIGURE 3

Uterine gland distribution in the postnatal uterus. Uterine gland morphology and volume along the anterior–posterior axis of the uterine horn. (a) P8, buds (blue circles), teardrops (green circles) (b) P11, buds, (blue squares), teardrops (green squares), elongated (red squares)

FIGURE 4

Identification of the uterine rail. (a, c, e, g) 3D reconstructions of the TROMA-1 stained prepubertal mouse uterine epithelium. Mesometrial views. Red dashed lines outline the uterine rail. (b, d, f, h) Optical cross-sections of fluorescent light sheet images of TROMA-1 stained uteri. Mesometrial side, top; arrowheads, uterine rail. (a, b) P0, (c, d) P8, (e, f) P11, and (g, h) P21. Scale bars for (a, c, e, g) = 150 μm. Scale bars for (b, d, f, h) = 50 μm

FIGURE 5

Uterine gland staging system and development. (a) Staging system for individual uterine glands. Stage 1 = bud. Stage 2 = teardrop. Stage 3 = elongated. Stage 4 = sinuous. Stage 5 = branched. (b) Correlation of gland formation initiation and morphogenesis with postnatal age