Perivascular cells support folliculogenesis in the developing ovary

Abstract

Supporting cells of the ovary, termed granulosa cells, are essential for ovarian differentiation and oogenesis by providing a nurturing environment for oocyte maintenance and maturation. Granulosa cells are specified in the fetal and perinatal ovary, and sufficient numbers of granulosa cells are critical for the establishment of follicles and the oocyte reserve. Identifying the cellular source from which granulosa cells and their progenitors are derived is an integral part of efforts to understand basic ovarian biology and the etiology of female infertility. In particular, the contribution of mesenchymal cells, especially perivascular cells, to ovarian development is poorly understood but is likely to be a source of new information regarding ovarian function. Here we have identified a cell population in the fetal ovary, which is a Nestin-expressing perivascular cell type. Using lineage tracing and ex vivo organ culture methods, we determined that perivascular cells are multipotent progenitors that contribute to granulosa, thecal, and pericyte cell lineages in the ovary. Maintenance of these progenitors is dependent on ovarian vasculature, likely reliant on endothelial-mesenchymal Notch signaling interactions. Depletion of Nestin⁺ progenitors resulted in a disruption of granulosa cell specification and in an increased number of germ cell cysts that fail to break down, leading to polyovular ovarian follicles. These findings highlight a cell population in the ovary and uncover a key role for vasculature in ovarian differentiation, which may lead to insights into the origins of female gonad dysgenesis and infertility.

Keywords: Nestin; blood vessels; ovarian follicle; ovary; perivascular cells.

Fig. 1.

Fetal ovarian Nestin⁺ cells are perivascular, pericyte-like cells. (A) E12.5 wild-type CD-1 fetal ovary. (B–E) E13.5 (B and D) and E18.5 (C and E) Nestin-CreER;Rosa-Tomato fetal ovaries exposed to 4-OHT at E12.5. Tomato is expressed in CSPG4⁺ (NG2⁺) perivascular cells (arrows) but not in ERG⁺ endothelial cells. (F and G) Flow cytometric analyses (F; n = 3 for E13.5 and E18.5) and EdU incorporation assays (G; n = 4 for E13.5 and n = 6 for E18.5) of E13.5 and E18.5 Nestin-CreER;Rosa-Tomato fetal ovaries exposed to 4-OHT at E12.5. Dashed lines indicate gonad–mesonephros border; A′–E′ are higher-magnification images of the boxed regions in A–E. (Scale bars: 50 μm.)

Fig. 2.

Initial labeling in Nestin-CreER–mediated lineage tracing of fetal and postnatal ovaries is targeted to perivascular cells with minimal granulosa cell labeling. (A and B) Long-term lineage-tracing of P60 Nestin-CreER;Rosa-Tomato ovaries exposed to 4-OHT at E12.5. Arrows indicate Tomato⁺ cells coexpressing FOXL2. (C–F) Nestin-CreER;Rosa-Tomato fetal and postnatal ovaries exposed to 4-OHT at E15.5 (C), E18.5 (D), P2 (E), and P4 (F). Images were taken 24 h after 4-OHT injection to determine initial Tomato reporter labeling. (Scale bars: 50 μm.) (G and H) Quantification (mean ± SD) of Tomato⁺ cell number (G; n = 5 for E16.5 and n = 4 for P1, P3, and P5) and percentage of Tomato⁺ cells coexpressing FOXL2 (H). Letters indicate statistical differences (P < 0.05).

Fig. 3.

Fetal and postnatal perivascular Nestin⁺ cells give rise to adult granulosa cells. (A–H) Long-term lineage tracing of fetal and postnatal Nestin⁺ cells in P30 (A, C, E, and G) and P60 (B, D, F, and H) Nestin-CreER;Rosa-Tomato ovaries exposed to 4-OHT at E15.5 (A and B), E18.5 (C and D), P2 (E and F), or P4 (G and H). E′–H′ are higher-magnification images of the boxed regions in E–H. Ovarian follicles are outlined by magenta dashed lines. (Scale bars: 50 μm.) (I) Graph shows percentage (mean ± SD) of primary, secondary, and antral follicles containing Tomato⁺ granulosa cells in P30 and P60 Nestin-CreER;Rosa-Tomato ovaries exposed to 4-OHT at E15.5 (n = 5 for P30 and P60), E18.5 (n = 7 for P30 and n = 6 for P60), P2 (n = 6 for P30 and n = 7 for P60), or P4 (n = 5 for P30 and n = 6 for P60). *P < 0.05, **P < 0.01.

Fig. 4.

Vasculature is required for maintenance of medullary perivascular Nestin⁺ cells. (A–C) Immunofluorescence images (A) and qRT-PCR analyses (mean ± SD) (B and C) of E12.5, E15.5, E18.5, or P2 wild-type CD-1 ovaries (n = 6) cultured ex vivo with DMSO (control) or VEGFR-TKI II (VEGFR inhibitor). *P < 0.05. (D) E18.5 Nestin-CreER;Rosa-Tomato fetal ovary after 24-h ex vivo exposure to 4-OHT. (E) Ex vivo vascular disruption of Nestin-CreER;Rosa-Tomato cultured fetal and postnatal ovaries after initial exposure to 4-OHT for 24 h. Dashed lines indicate gonad–mesonephros border or gonad boundary. (Scale bars: 50 μm.)

Fig. 5.

Vascular disruption results in differentiation of Nestin⁺ cells into cortical pregranulosa cells. Immunofluorescence images (A–C) and qRT-PCR analyses (mean ± SD) (D) of E15.5 (A), E18.5 (B), or P2 (C) Nestin-CreER;Rosa-Tomato ovaries cultured ex vivo with DMSO (control) or VEGFR-TKI II. Arrows throughout indicate Tomato⁺/FOXL2⁺ cells. Dashed outlines in A–C indicate gonad boundary. The Rightmost image in each panel is a higher-magnification image of the boxed region in the Leftmost image. (Scale bars: 50 μm.) (D) Fraction of Tomato expression overlapping with FOXL2 in different stages of ex vivo cultured ovaries (n = 3). *P < 0.05, **P < 0.01.

Fig. 6.

Vasculature is essential for medullary Notch activity in fetal and postnatal ovaries. Immunofluorescence images (A–D) and qRT-PCR analyses (mean ± SD) (E) of E12.5 (A), E15.5 (B), E18.5 (C), or P2 (D) CBF:H2B-Venus ovaries (Notch signaling reporter) cultured ex vivo with DMSO (control) or VEGFR-TKI II. Arrows throughout indicate Venus⁺ cells in the gonad or mesonephros. The image on the Right in each panel is a higher-magnification image of the boxed region in the Left image. Dashed lines in A and B indicate gonad–mesonephros border. (Scale bars: 50 μm.) (E) qRT-PCR analyses showing fold change in Notch target gene (Hes1, Hes5, Hey1, and Heyl) mRNA levels after disruption of vasculature in E12.5, E15.5, E18.5, or P2 cultured CD-1 ovaries (n = 3). *P < 0.05.

Fig. 7.

Disruption of Notch signaling induces differentiation of Nestin-derived cells into pregranulosa cells. (A–F) qRT-PCR analyses (mean ± SD) (A, E, and F) (n = 3 to 4 for A; n = 6 for E and F) and immunofluorescence images (B–D) of E15.5 (B), E18.5 (C), or P2 (D) CD-1 ovaries cultured ex vivo with DMSO (control) or DAPT (Notch signaling inhibitor). (G) Ex vivo culture of E15.5, E18.5, or P2 Nestin-CreER;Rosa-Tomato fetal and postnatal ovaries. Right image for @E18.5 samples is a higher-magnification image of the boxed region in the Left image. Dashed lines throughout indicate gonad outline or border. (Scale bars: 50 μm.) (H) Fraction of Tomato expression overlapping with FOXL2 (mean ± SD) in different stages of ex vivo cultured ovaries (n = 3). *P < 0.05, ***P < 0.001.

Fig. 8.

Conditional disruption of Notch signaling specifically in Nestin⁺ cells results in differentiation of Nestin-derived cells into pregranulosa cells. Immunofluorescence images of P2 (A) and P4 (C) Nestin-CreER;Rosa-Tomato;Rbpj-flox/+ heterozygous control (Top) and Nestin-CreER;Rosa-Tomato;Rbpj-flox/flox (Bottom) postnatal ovaries exposed to 4-OHT at E18.5 and P2, respectively. (B) Graph shows percentage of FOXL2-expressing cells among Tomato⁺ cells (mean ± SD) in P2 control (n = 6) and Rbpj conditionally deleted ovaries (n = 3) exposed to 4-OHT at E18.5. (Scale bars: 50 μm.) ***P < 0.001.

Fig. 9.

Postnatal Nestin-derived cells are required for folliculogenesis. (A) Cartoon depicting experimental strategy for ablating postnatal Nestin⁺ cells in Nestin-CreER;Rosa-Tomato;Rosa-DTA postnatal ovaries via exposure to 4-OHT at P2 and P4. (B–G) Hematoxylin staining (B and E), quantification of polyovular follicles (C and F), and follicle counts at different developmental stages (D and G) (mean ± SD) in Nestin-CreER;Rosa-Tomato;Rosa-DTA P7 (B–D) and P21 (E–G) ovaries. Magenta outlines denote polyovular follicles. n = 4 for each genotype and stage. *P < 0.05, **P < 0.01. (Thin scale bars: 50 μm; thick scale bars: 5 μm.) (H) Cartoon model depicting the dynamic marker expression pattern and function of Notch-signaling–regulated perivascular Nestin⁺ cells in the ovary.