Increased DNA Damage and Repair Deficiency in Granulosa Cells Are Associated with Ovarian

Supplemental data

Increased DNA damage and repair deficiency in granulosa cells are associated with ovarian aging in rhesus monkey

Dongdong Zhang et al.

Supplemental Figures S1-8

Figure S1.Histology of ovarian morphology by H&E from young (3-4 years), middle-age (7-8 years), and old monkeys (18-19 years) (upper and lower panels from two monkeys). PF, primordial & primaryfollicles; SF, secondary follicles; AnF, antral follicles; AtF, Atretic follicles. Antral follicles possess small areas of follicular fluid (antrum) or a single large antrum. Atretic follicles are those with collapse of granulosa cells but without clear cavity. Scale bar=100μm; Note, healthy antral follicles are rarely found in old monkey ovaries.

Figure S2.Immunocytochemistry of markers for DSBs and telomere in somatic cells at the cortex in adult monkey ovaries by co-immunostaining of H2AX and TRF2. (A) Representative morphology of two adjacent cortex somatic cells in the ovaries of monkeys at various reproductive ages. (B) Number of H2AX foci in cortex somatic cells. (C) Number of H2AX and TRF2 co-localization foci in cortex somatic cells.Scale bar=10μm; Error bars show mean+SEM. *P<0.05. n= number of cells counted.

Figure S3.Analysis of double strand breaks (DSBs) by immunostaining and epi-fluorescence microscopy of DNA DSB marker H2AX in monkey oocytes. (A) Representative images showing H2AX immunofluorescence and foci in the oocytes within primordial＆primary follicles in the ovaries from young (Y) and middle-age (M) monkeys. (B) Average number of H2AX foci per oocyte GV nucleus by 5 µm section of primordial＆primary follicles. (C) Representative images showing H2AX immunofluorescence and foci in the oocytes of secondary follicles. The white broken circles indicate area of a GV nucleus. (D) Average number of H2AX foci per nuclear section in GV oocytes of secondary follicles. Scale bar=10 µm; Error bars show mean+SEM. *P<0.05. n= number of GV nuclei counted.

Figure S4. BRCA1 immunofluorescence and foci in Hela cells.

(A) Two representative images showing BRAC1 immunofluorescence and foci in Hela cells. (B) Representative image showing no BRAC1 immunofluorescence and foci in Hela cells without using the antibody served as negative control. Scale bar=20 µm. Images for BRCA1 red fluorescence were taken using 63X oil lens with the same exposure time at 600ms using Zeiss Axio Imager Z1.

Figure S5.BRCA1 brownish staining detected by immunohistochemistry in monkey ovaries by 3,3-diaminobenzidine (DAB) reaction. Sections of ovaries of monkeys at various reproductive ages were immunoreactively stained by BRCA1 antibody, followed by exposure to DAB for the same short period of time. Sections without incubation with BRCA1 antibody did not show brownish staining following reaction with DAB, served as negative control (data not shown). Young (Y), monkey at the age of 3-4 years; Middle-age (M), monkey at 7-8 years; Old (O), reproductive aging monkeys at 18-19 years. Original magnification, 630X.

Figure S6.Immunocytochemistry by immunostaining and epi-fluorescence microscopy of DSB repair related gene BRCA1 in granulosa cells of follicles at different developmental stages in the ovaries from young (Y), middle-age (M) and old (O) monkeys. “+”, BRCA1 antibody was added; “-”, BRCA1 antibody was not added, served as negative controls, which showed only background fluorescence without BRCA1 specific foci. Scale bar=10 μm.

Figure S7.Analysis of BRCA1 foci revealed byimmunofluorescence microscopy in granulosa cells of various follicles.(A) Representative images showing BRAC1 immunofluorescence and foci in granulosa cells of the primordial＆primary follicles in the ovaries from young (Y), middle-age (M) and old (O) monkeys. (B) Number of BRCA1 foci per nuclear section in granulosa cells of primordial＆primary follicles.(C) Representative images showing BRAC1 immunofluorescence and foci in the secondary follicles. (D) Number of BRCA1 foci per nuclear section in granulosa cells of secondary follicles. (E,F) Immunocytochemistry and count of BRCA1 foci in granulosa cells of antralfollicles in the ovaries of young and middle-age monkeys.Scale bar=10 μm; Error bars show mean+SEM. *P<0.05; **P<0.01; ***P<0.001. n= number of cells counted.

Figure S8.Relative telomere length expressed as T/S ratio measured by quantitative real-time PCR method. Bars indicate mean±s.d. (n=3).

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