Suppression of Notch signaling in the neonatal mouse ovary decreases primordial follicle formation

Abstract

Notch signaling directs cell fate during embryogenesis by influencing cell proliferation, differentiation, and apoptosis. Notch genes are expressed in the adult mouse ovary, and roles for Notch in regulating folliculogenesis are beginning to emerge from mouse genetic models. We investigated how Notch signaling might influence the formation of primordial follicles. Follicle assembly takes place when germ cell syncytia within the ovary break down and germ cells are encapsulated by pregranulosa cells. In the mouse, this occurs during the first 4-5 d of postnatal life. The expression of Notch family genes in the neonatal mouse ovary was determined through RT-PCR measurements. Jagged1, Notch2, and Hes1 transcripts were the most abundantly expressed ligand, receptor, and target gene, respectively. Jagged1 and Hey2 mRNAs were up-regulated over the period of follicle formation. Localization studies demonstrated that JAGGED1 is expressed in germ cells prior to follicle assembly and in the oocytes of primordial follicles. Pregranulosa cells that surround germ cell nests express HES1. In addition, pregranulosa cells of primordial follicles expressed NOTCH2 and Hey2 mRNA. We used an ex vivo ovary culture system to assess the requirement for Notch signaling during early follicle development. Newborn ovaries cultured in the presence of gamma-secretase inhibitors, compounds that attenuate Notch signaling, had a marked reduction in primordial follicles compared with vehicle-treated ovaries, and there was a corresponding increase in germ cells that remained within nests. These data support a functional role for Notch signaling in regulating primordial follicle formation.

Figure 1

Notch family gene expression in the neonatal mouse ovary. A, RT-PCR was performed to detect Notch receptor, ligand, and target gene transcripts in ovaries from d 3 CD-1 mice. Primers for ribosomal protein RPL19 were used as a control. A complete list of primer sequences can be found in supplemental Table 1. Notch2 primers were used for the negative control reverse transcription reaction. B, Real-time PCR was used to examine the regulation of selected Notch family transcripts during postnatal ovarian development. The second y-axis represents relative mRNA values for Hey2. The comparative Ct method (ΔΔCt) was used for relative quantification. Genes were normalized to RPL19, and the graph shows changes for each gene relative to d 0. The inset shows mRNA values for Notch2 (N2), Notch3 (N3), Jagged1 (J1), Jagged2 (J2), Hes1 (Hs1), and Hey2 (Hy2) at d 0, normalized to RPL19. The graph represents the average of samples from three to five experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2

NOTCH2, JAGGED1, and HES1 localization in the neonatal mouse ovary. In the newborn ovary, NOTCH2 and HES1 were expressed in pregranulosa cells of germ cell nests (A and E, arrows), whereas the Notch ligand JAGGED1 was expressed in germ cells of germ cell nests (C, arrows). Three days into postnatal development, the pregranulosa cells of primordial follicles stained positively for NOTCH2 and HES1 (B and F, arrows), and oocytes surrounded by a mixture of squamous and cuboidal somatic cells (F, double arrow) also expressed HES1. By d 3, JAGGED1 expression was maintained in oocytes of primordial follicles (D, arrows). Experiments were repeated three times and representative images are shown. Scale bars, 25 μm. GR, Pregranulosa cells; GE, germ cells; Oo, oocytes.

Figure 3

In situ hybridization for Hey2 mRNA in the d 3 ovary. Ovary sections were incubated with either an antisense Hey2 (A) or sense Hey2 (B) probe. Hey2 transcripts were expressed in pregranulosa cells (A, arrows) and oocytes (A, double arrow) of primordial follicles. Scale bars, 25 μm. GR, Pregranulosa cells; Oo, oocytes.

Figure 4

Cellular dynamics in cultured and in vivo ovaries. The TUNEL assay was used to determine the distribution of apoptotic cells between isolated (A) and cultured ovaries (B). Newborn mouse ovaries were cultured for 4 d. Immunohistochemistry for proliferating cell nuclear antigen (PCNA) was used to assess cell proliferation in isolated (C) and cultured ovaries (D). The concentration of insulin used in these ovary culture studies was 50 μg/ml. Scale bars, 40 μm (A and B); 50 μm (C and D).

Figure 5

γ-Secretase inhibitor efficacy in ovary cultures. Real-time PCR was used to measure mRNA expression of selected genes in d 0 ovaries treated with DMSO or DAPT for 1 d (A) or 4 d (B) (n = 6–8/group). The concentration of insulin used in these ovary culture experiments was 10 μg/ml. Relative mRNA values in these studies were normalized to RPL19. The second y-axis corresponds to relative mRNA values for GAPDH. The graph represents the average of samples from three experiments. *, P < 0.05; **, P < 0.005.

Figure 6

Phenotypes of ovary cultures treated with vehicle and DAPT. Newborn mouse ovaries cultured for 4 d in media containing 0.2% DMSO (A) or 20 μm DAPT (B) were fixed and then H+E stained. Germ cell nests are indicated by black boundaries. C and D, Enlarged images of the black rectangles in A and B. E, Follicle populations in DMSO- (n = 4) and DAPT (n = 5)-treated ovaries were quantified. The concentration of insulin used in these studies was 1 μg/ml. The graph represents average follicle counts from ovaries cultured in three independent experiments. Scale bars, 50 μm. *, P < 0.05.

Figure 7

Ovary cultures treated with a second γ-secretase inhibitor, L-685,458. Ovaries cultured for 4 d in media containing 0.2% DMSO (A) or 10 μm L-685,458 (B) were fixed and then H+E stained. Germ cell nests are indicated by black boundaries. C and D, Enlarged images of the black rectangles in A and B. E, Follicle populations in ovaries treated with DMSO (n = 5) and L-685,458 (n = 7) were counted. The concentration of insulin used in these studies was 50 μg/ml. The graph represents average follicle counts from ovaries cultured in four independent experiments. Scale bars, 50 μm. *, P < 0.005.

Figure 8

Examination of apoptosis in vehicle and DAPT-treated ovaries. Ovaries were cultured for 1, 2, or 4 d with DMSO or DAPT. A, A TUNEL-stained tissue section from an ovary treated with DAPT for 1 d. B, Enlarged image of the white rectangle in A showing apoptotic pregranulosa cells (arrows) and an apoptotic germ cell (double arrow). C, The graph represents the average number of TUNEL-positive cells in one tissue section from three different control and inhibitor-treated ovaries at each time point. The concentration of insulin used in these studies was 10 μg/ml. Scale bars, 40 μm. *, P < 0.05. GR, Pregranulosa cells; GE, germ cells.