www.sciencemag.org/content/343/6170/533/suppl/DC1

Supplementary Materials for

Reversal of Female Infertility by Chk2 Ablation Reveals the Oocyte DNA Damage Checkpoint Pathway

Ewelina Bolcun-Filas, Vera D. Rinaldi, Michelle E. White, John C. Schimenti*

*Corresponding author. E-mail: jcs92@cornell.edu

Published 31 January 2014, Science 343, 533 (2014) DOI: 10.1126/science.1247671

This PDF file includes:

Materials and Methods Figs. S1 to S8 References

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Materials and Methods

Mice

The alleles used in this study and their genetic backgrounds were: Trip13 Gt(RRB047)Byg

(C57Bl/6J)(abbreviated as Trip13Gt in text) (12), Dmc1 tm1Jcs (C57Bl/6J)(11), Atmtm1Awb

(C3H/HeJ)(25), Trp53tm1Ty (C3H/HeJ) (26), Chk2 tm1Mak (C3H/HeJ)(27)and a TA-domain specific

deletion of p63 made in the lab of Alea Mills (18). This last mutant does not have a formal allele

name. The original paper describes its construction, which contained loxP sites flanking the TA

domain encoded by exons 2 and 3, and was referred to as Trp63F(18). The allele used here is

derived from that allele, but exons 2 and 3 were previously removed by Cre-deletion. Herein we

described the allele as TAp63- (129S7/SvEvBrd × C57BL/6 mixed background). Comparisons of

compound mutants and controls utilized littermates whenever possible. All experiments were

done with at least three mice per experimental group, unless indicated otherwise.

Fertility tests

Control and experimental females (2-6 months old) were housed with fertile males. A

female was considered fertile if she gave multiple births. Litter sizes were determined by

counting pups on the day of birth. Embryo loss during pregnancy was assessed by comparing the

numbers of implantation sites at 12.5 days post coitum (dpc) to the number of corpora lutea (CL)

in the ovary, the latter of which indicates the numbers of ovulated oocytes. Briefly, females were

mated to WT males and the presence of a copulation plug in the morning was recorded as 0.5

dpc. At 12.5 dpc, females were sacrificed and the ovaries and uterus were dissected. Numbers of

implantation sites, normal and abnormal embryos and CL were scored and counted. Graphing

and statistical analyses were performed with GraphPad Prism5.

Histology and follicle quantification

Ovaries were fixed in Bouin’s, embedded in paraffin, serially sectioned at 5μm and

stained by hematoxylin and eosin. Follicle quantification was performed as described (28).

Every fifth section was examined for the presence of the following classes of oocytes/follicles:

primordial, primary, secondary, preantral and antral. For quantitative analyses, they were

grouped into two classes: 1) primordial and 2) everything else (primary through antral). Graphs

and statistical analysis was performed with GraphPad Prism5. Follicle number comparisons

between genotypes were evaluated using the non-parametric two-tailed Mann-Whitney test.

Immunohistology

Immunolabeling of surface-spread newborn oocytes was performed as described (29).

Germinal Vesicle (GV) stage oocytes were isolated and immunostained as described (30).

Primary antibodies used in this study: rabbit anti-SYCP3 (1:500, Abcam); mouse anti-γH2AX

(1:500, JBW301 Upstate Biotechnology); mouse anti-p63 4A4 (1:250, Novus Biologicals); and

rabbit anti-MVH (1:500, Abcam). Secondary antibodies: Alexa Fluor488 and Alexa Fluor594

(Molecular Probes). DNA was stained with DAPI. Images were acquired with an Olympus BX51

microscope and processed with MagnaFire 2.0.

Animal irradiation

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Five day old females were exposed to a single dose of ionizing radiation (0.45Gy) in a 137cesium irradiator with rotating turntable. They were monitored daily for 2 weeks after

radiation and weekly thereafter.

Ovary irradiation for oocyte survival analysis

Ovaries from one week old pups were dissected, placed into Millicell-CM culture inset

chambers and incubated in MEM media (MEM+ 10%FBS, 25mM Hepes, pen-strep) at 37 °C

under an atmosphere containing 5% CO2. Ovaries from each genotype were exposed to 0.45 Gy

of ionizing radiation and cultured for 7 days. Irradiated and non-irradiated cultured ovaries were

fixed in 4% paraformaldehyde and embedded in paraffin for immunohistochemistry. For germ

cell comparisons, 5m ovary sections were immunostained with anti-MVH and anti-p63 using

standard methods.

Western Blot analysis of protein phosphorylation

Newborn WT and Chk2 -/- ovaries were exposed to 3Gy of ionizing radiation as described

above and proteins were extracted 2 hrs post irradiation. For mutants, ovaries from all newborn

females in the litter were dissected and individually frozen while genotyping was performed.

Proteins from ovaries of selected genotypes (6-12 ovaries) were extracted with lysis buffer

containing 1% NP40, 1% SDS, 20mM TrisCl (pH8.1), 150mM NaCl, 1mM EDTA, 10%

Glycerol, Protease Inhibitors (Complete Mini-Roche#11836153001), Phosphoprotease Inhibitors

(PhosSTOP-Roche# 04906845001), 2.5 mM Sodium Pyrophosphate, 1 mM -

glycerophosphate, 1 mM Na4VO3. Proteins were resolved in 10% acrylamide, transferred to

nitrocellulose membranes (BioRad) that were blocked with 5% nonfat milk. To assess protein

phosphorylation, membranes were probed with mouse anti-p63 4A4 (1:500, Novus Biologicals);

rabbit anti-p53 (1:300, Cell Signaling); mouse anti-γH2AX (1:500, JBW301 Upstate

Biotechnology) and rabbit anti-MVH (1:1000, Abcam).

Identification and functional analysis of a p63 phosphorylation site in CHK2

We searched the p63 amino acid sequence (NP_001120731.1) for presence of the CHK2

consensus phosphorylation site LxRxxS/T (31) and found that it contained LLRTPS, with the

Serine being at position 621. A plasmid bearing FLAG-tagged TAp63alpha (from David

Sidransky, Addgene plasmid 27008) was mutagenized to change position 621 to Alanine using

the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). Wild type

S621 and mutant S621A TAp63alpha-FLAG plasmids were transfected into HeLa cells using the

TransIT®-LT1 Transfection Reagent (Mirus). Transfected cells were exposed to 0 or 3Gy of

ionizing radiation and protein extracts were collected 2hrs post radiation. Extracts from control

and irradiated cells were analyzed by Western blot with anti-FLAG (Cell Signaling) and anti-

CHK2 (Millipore) antibodies.

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Fig. S1. Timing of DNA damage and asynapsis checkpoints in oocytes as observed in

various meiotic mutants.

5

Fig. S2. Quantification of follicle types in 3 weeks old single and double mutants.

“Other follicles” include primary, secondary, preantral and antral. Statistical analysis was a non-

parametric two-tailed Mann-Whitney test.

6

Fig. S3 Ovarian histology of 2 month old double mutants.

Permanent (long term) rescue of resting oocyte pool occurred only in Trip13Gt/Gt Chk2-/- mice

(see inset showing abundant primordial follicles; this contrasts with Dmc1-/- Chk2-/- ovaries, in

which only more developed follicles can be seen, the earliest being primary, not primordial).

Arrowheads indicate primordial follicles. An- Antral follicle, 1º- primary follicle, 2º- secondary

follicle, CL- Corpus Luteum. Scale bar = 200 m.

7

Fig. S4 Quantity and viability of oocytes and embryos produced by Trip13Gt/Gt Chk2-/-

(DKO) females mated to WT males.

The “control” in this situation are Trip13Gt/+ Chk2-/-. CL = Corpora Lutea; this represents the

number of ovulated oocytes. Statistical analysis was a non-parametric two-tailed Mann-Whitney

test.

8

Fig. S5 Assessment of DNA damage in rescued oocytes.

(A) By 2 months of age, levels of DSBs in rescued oocytes (positive for the germ cell protein

MVH) are indistinguishable from controls (left panel). Occasional H2AX positive oocytes are

present in both, and possibly reflect atretic follicles. Pd, primordial; Pr, primary; Pa; preantral

follicles. (B) Shown are nuclei of preovulatory GV stage oocytes. No marks of DSBs (H2AX)

were evident in Trip13Gt/Gt Chk2-/- or Trip13Gt/Gt mutant GV stage oocytes collected at 3 weeks of

age, suggesting completed DSB repair. Arrowhead indicates nucleus. Green color is

autofluorescence.

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Fig. S6 Proposed DNA damage response pathway in mouse oocytes.

Circled "P" denotes phosphorylation. Dashed arrow between p53 and p63 represents a regulatory

relationship discussed in text, whereby the absence of one stimulates the use of the other in either

pachytene or resting oocytes.

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Fig. S7 Chk2 deficiency partially rescues Atm-/- mutant oocytes.

H&E stained mouse ovaries of indicated genotypes. Scale bar = 200 m. The primordial oocyte

pool is depleted by 2 months of age (bottom panel).

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Fig. S8 DSB repair-defective spermatocytes are not noticeably rescued by p53 or Chk2

mutations.

Shown are H&E-stained mouse testis sections of the indicated genotypes. As in Trip13Gt/Gt single

mutants, there is no progression of spermatocytes past the pachytene stage when either

checkpoint gene is removed. Scale bar = 100 m.

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