Follicle dynamics and global organization in the intact mouse ovary

Abstract

Quantitative analysis of tissues and organs can reveal large-scale patterning as well as the impact of perturbations and aging on biological architecture. Here we develop tools for imaging of single cells in intact organs and computational approaches to assess spatial relationships in 3D. In the mouse ovary, we use nuclear volume of the oocyte to read out quiescence or growth of oocyte-somatic cell units known as follicles. This in-ovary quantification of non-growing follicle dynamics from neonate to adult fits a mathematical function, which corroborates the model of fixed oocyte reserve. Mapping approaches show that radial organization of folliculogenesis established in the newborn ovary is preserved through adulthood. By contrast, inter-follicle clustering increases during aging with different dynamics depending on size. These broadly applicable tools can reveal high dimensional phenotypes and age-related architectural changes in other organs. In the adult mouse pancreas, we find stochastic radial organization of the islets of Langerhans but evidence for localized interactions among the smallest islets.

Keywords: Confocal imaging; Islet of Langerhans; Ovary; Pancreas; Primordial follicle; Whole tissue labeling.

Figure 1

Oocyte nuclear markers enable spatial resolution of follicles in wholemount neonatal mouse ovaries. Extended projections of Nobox immunostaining in an intact PD1 ovary (A). Optical z stack slices through a PD1 ovary stained with Nobox (red) and GCNA (blue) show surface localization of the GCNA+ cells and exclusive Nobox⁺ cells in the core (B). Extended z-stack projection of Nobox staining in an intact PD5 ovary (C). Selection of Nobox⁺ objects is indicated by separate colors, inset. Frequency distribution of Nobox volumes compared with GCNA at PD1 (D) shows cutoff for PFs at 3000 µm³. Comparison of Nobox frequency distributions reveals a stepwise increase of largest object sizes from birth to PD5 to PD7 by the lengthening of the right tail, and a concomitant loss of smallest objects (E). Scale bars represent 150 µm.

Figure 2

WM imaging of oocyte nuclei in prepubertal and adult ovaries reveals similar growth dynamics. In 3D reconstruction of a PD21 ovary (A), a range of object sizes is visible and similarly at 6-weeks (B) nonspecific staining occurs in vasculature of the hylem (arrowhead). Nobox penetrates the adult ovary, as shown in optical z stack slices at 24 weeks (C). Nobox profiles are largely similar between ovaries of young (12 week) and old (24 week) adults (D). One week following cyclophosphamide treatment, the most significant losses occur in large Nobox⁺ objects (>10,000 µm³), although small objects are also decreased. Arrows in all graphs indicate size cutoffs at 3000 and 10,000 µm³ (E). Scale bars represent 150 µm in panels A–B, and 600 µm in C.

Figure 3

Quantification of primordial follicles in WM mouse ovaries is consistent with a finite ovarian reserve. A, Nobox-positive objects in juvenile and adult ovaries are tabulated and represented graphically (B), with relative proportion of each size class in the inset. Volume cutoffs for small, medium and large classes of objects are shown. C, The mean number of small Nobox⁺ objects, representing PFs, is plotted versus time and fit to a logarithmic (grey) and power function (black). D, Examination of PD7 ovaries with the proliferation marker pHH3 (blue) reveals very rare colabeling with Nobox (red; 5/17,972 in n=6 ovaries). Scale bar, 20 µm.

Figure 4

Follicle spatial dynamics in neonatal ovaries persist through adulthood. Location of small Nobox⁺ objects (<3000 µm³, gold), medium-sized (3000–10,000 µm³, brown), and large (>10,000 µm³, turquoise) are shown in 3D opacity projections of ovaries at PD5 (A), 3 weeks (C), 6 weeks (E), and 24 weeks (G). For each ovary, measurements from the center of each Nobox object to the ovary surface (B, D, F, H) are shown with respect to object volume. At all stages, small oocyte nuclei (gold) skew toward the surface, whereas medium sized nuclei most frequently reside 50–150 µm from the surface. Median depths for each size class are indicated by grey bars. Exemplary aggregations of objects in 3D are indicated in panels E and G by arrowheads of the corresponding color. Scale bars represent approximately 150 µm.

Figure 5

Spatial analysis of follicular clustering with age and follicle size. The distribution of differently-sized Nobox objects was assessed using Ripley’s K-function to identify second-order properties. The K(d)−E(K(d)) curves depict deviations of the follicle distribution (K(d)) from randomness (E(K(d)) over a given distance (d) between Nobox⁺ object centroids. Positive values indicate clustering while negative values indicate dispersion. The change in distribution of small, medium, and large oocyte nuclei (A, B, C) was evaluated as ovaries matured from 1–24 weeks. Objects of all sizes displayed an increasing trend towards clustering with age. The comparative distributions of differently-sized follicles at a given stage was also investigated at 3, 6, and 24 weeks (D, E, F). Large Nobox⁺ objects are comparatively more dispersed than medium and small through 6 weeks of age, but become similarly aggregated by 24 weeks. Maximum diameters for primordial, primary and secondary follicles are indicated by arrowheads for comparison.

Figure 6

3D analysis of islets of Langerhans in the adult mouse pancreas. Islets were visualized and selected in the pancreas by Nkx6.1 wholemount staining, as shown in extended projection (A). Nkx6.1 volume profiles are similar between dorsal and ventral pancreas (B), with breaks between small, medium and large classes shown by arrows. Distribution of Nkx6.1 object volume versus 3D shape factor (a measure of sphericity) shows a similar trend toward elongation with increasing size for both dorsal and ventral pancreas (C). Radial distance analysis of Nkx6.1⁺ objects from a surface defined by the same objects reveals an absence of correlation between volume and depth (D). Clustering analysis of Nkx6.1⁺ objects in the entire pancreas finds no difference between the largest class and random distribution, but a departure of the small and medium object relationships K(d) from the expected random distribution E(K(d)) (E). The ratio of these distributions reveals a maximal clustering behavior of small objects at a distance of 40–80 µm, which exceeds the maximal inter-centroid distance of 34 µm (grey arrowhead); a smaller magnitude of attraction occurs between medium size Nkx6.1 objects at a distance between their minimum (grey arrowhead) and maximum (green arrowhead) adjacent distance (F). Scale bar represents 1 mm.