A Roadmap for Three-Dimensional Analysis of the Intact Mouse Ovary

Abstract

Recent advances in tissue clearing methodologies have enabled three-dimensional (3D) visualization of the ovary and, consequently, in-depth exploration of the dynamic changes occurring at the single-cell level. Here we describe methods for whole-mount immunofluorescence, clearing, imaging, and analysis of whole ovarian tissue in 3D throughout murine development and aging.

Keywords: 3D analysis; Imaging; Ovary; Tissue clearing; Whole-mount immunofluorescence.

Figure 1.

Decision flow chart for choosing the optimal protocol to whole-mount stain and clear the mouse ovary. Washing steps and the final clearing step are ideal pause points if necessary. Steps in bold should be kept consistent to obtain comparable data. *: iDisco clearing is more compatible when the endogenous signal is high. δ: While both BABB and iDisco are effective at this timepoint, BABB is more time-efficient.

Figure 2.

Identifying the feature size (XY diameter) of NOBOX+ germ cells in postnatal day 16 (PN16) ovary. A. Click the section view tab (bordered by a yellow rectangle) to visualize individual z stacks. Based on the stage of the ovary, the size of objects might vary due to different stages of oocyte development (yellow arrows showing growing and non-growing follicles). Choose the line option (white arrow) under the measure tab on the right-hand side. B and C. Draw a line from one margin to another to measure XY diameter (yellow arrow), the distance value is shown under the measure tab (white arrow).

Figure 3.

Segmentation efficiency of PN16 ovary imaged with 10X and 25X objectives. The number of NOBOX+ germ cells (in magenta) of the same ovary were analyzed after imaging with the 10X (on the left) and the 25X objective (on the right). Big gray and small white circles demonstrate accurately picked growing and non-growing follicles, respectively. The table shows raw germ cell counts in the same sample after imaging with the 10X and 25X objectives and applying the same analysis pipeline. Please note that the difference in the number of non-growing follicles that are closely localized is more prominent than growing follicles. Arrow indicates the region where more non-growing germ cells were picked with 25X objective compared to 10X.

Figure 4.

Spatial analysis of germ cell distribution using the custom MATLAB script. Following whole-mount staining of the E16.5 ovary with TRA98 (in gray) and imaging with a confocal microscope, a surface is created on the ovary, and the mesonephros is removed by masking the TRA98 staining channel. Spots that represent individual TRA98+ germ cells are created and coordinates of the spots are used for downstream analysis. The ovary is divided into seven segments based on their medial (bins 3–5) - lateral (bins 1–2 and 6–7) and anterior (from bin 1) - posterior (to bin 7) distributions, respectively. Total germ cell numbers of each segment are graphed.