www.sciencetranslationalmedicine.

org/cgi/content/full/5/185/185ra62/DC1

Supplementary Materials for

Cyclophosphamide Triggers Follicle Activation and “Burnout”; AS101
Prevents Follicle Loss and Preserves Fertility
Lital Kalich-Philosoph, Hadassa Roness, Alon Carmely, Michal Fishel-Bartal, Hagai
Ligumsky, Shoshana Paglin, Ido Wolf, Hannah Kanety, Benjamin Sredni,*
Dror Meirow*
*Corresponding author. E-mail: meirow@post.tau.ac.il

Published 15 May 2013, Sci. Transl. Med. 5, 185ra62 (2013)

DOI: 10.1126/scitranslmed.3005402

The PDF file includes:

Fig. S1. The PI3K/PTEN/Akt signaling pathway regulates primordial follicle

suppression and activation.
Fig. S2. Cleaved caspase-3 staining is increased after Cy treatment.
Fig. S3. Cy treatment stimulates proliferation, but not apoptosis, of primordial
follicles, and AS101 reverses this effect.
Fig. S4. Female mice in each subgroup of the mating study have similar weights
and plasma SAA concentrations.
Fig. S5. AS101 increases MCF-7 and MDA-MB-231 cell sensitivity to
chemotherapy.
Fig. S6. All positive controls showed appropriate staining.
Table S1. Cy decreases the incidence of pregnancy in mice, and AS101 rescues it.
SUPPLEMENTARY MATERIALS

Supplementary Figure 1

The PI3K/PTEN/Akt signaling pathway regulates primordial follicle suppression and

activation.

The PI3K/PTEN/Akt signaling pathway is responsible for control of follicle quiescence and
activation. Equilibrium within the pathway is regulated by PTEN, and is essential for maintaining a
state of dormancy in the primordial follicle reserve. Changes in signal transduction elements can
trigger increased activation of a phosphorylation cascade that results in activation of the primordial
follicle (PMF). Phosphorylation of molecules that appear in green, such as Akt, mTOR and rpS6,
causes activation of primordial follicles, while those in red, such as PTEN, TSC and FOXO3,
maintain follicle dormancy.
Supplementary Figure 2.

Cleaved caspase-3 staining is increased after Cy treatment.

Ovaries from adult (8-week-old) mice were removed 24 hours after treatment with 150 mg/kg Cy or
PBS, then stained for apoptosis with cleaved caspase 3 antibody. Staining was observed in primary,
secondary and pre-antral follicles, but not in primordial follicles. Sections of spleen from mice
treated with Cy were used as positive controls (Figure S6). A representative section from each group
is shown. The arrow indicates staining in granulosa cells and oocyte of an early growing follicle.
Scale bar a, c = 200 µm; b = 100 µm; d, e = 50 µm.
Supplementary Figure 3.

Cy treatment stimulates proliferation, but not apoptosis, of primordial follicles, and AS101
reverses this effect.

A) Ovaries from adult (8-week-old) mice were removed 24 hours after treatment with 150 mg/kg Cy
and subjected to TUNEL staining, which showed widespread apoptotic death of granulosa cells,
both in secondary and large growing follicles. No apoptosis was detected in dormant primordial
follicles. Co-treatment with AS101 decreased the apoptosis in growing follicle populations. Red
circle indicates unstained primordial follicle. Scale bar a, b, c, d, e, j, m, n = 100 µm; h, i = 50 µm; f,
g, k, l = 25 µm.
B) Ki67 staining of ovaries removed 24 and 72 hours after treatment with 150 mg/kg Cy with or
without AS101 co-administration shows that treatment with AS101 decreased the extent of
proliferation in the primordial and primary follicles after Cy. More granulosa cells stained positive
for growth in Cy-treated ovaries than in AS101+Cy ovaries. At 72 hours after treatment, more
granulosa cells in primary follicles in the Cy treatment group stained positive for growth, compared
with the AS101+Cy group. Red circles indicate unstained primordial follicles. Scale bar a = 200
µm; e = 100 µm; b, c, f, g = 25 µm; d, h = 20 µm.
Supplementary Figure 4

Female mice in each subgroup of the mating study have similar weights and plasma SAA
concentrations.

A) The weights of mice in each group were not statistically different.

B) Serum amyloid A (SAA) concentrations were determined in plasma samples taken from mice at
the time of death. There was no statistical difference between SAA levels in mice from the various
treatment groups.
Supplementary Figure 5

AS101 increases MCF-7 and MDA-MB-231 cell sensitivity to chemotherapy.

A,B) MCF-7 and C,D) MDA-MB-231 human breast cancer cell lines were treated with increasing
concentrations of phosphoramide mustard CS, with or without AS101 (2.5 µg/ml and 5 µg/ml for
MCF-7 and for MDA-MB-231 cells). MTT assay was conducted at 72 hrs to assess cell viability.
Data represent the mean ± SEM of at least three independent experiments, performed in
quadruplicate.
Supplementary Figure 6

All positive controls showed appropriate staining.

Samples of positive controls used for immunohistochemical and immunofluorescent staining

specific to each antibody are shown above:
• Caspase 3 - sections of spleen from mice treated with Cy
• TUNEL - rat mammary gland, as supplied by the kit (cross reactive with mice)
• Ki67 - mouse intestine
• pAkt, pMTOR and pFOXO3a - estrogen (ER) and progesterone (PgR) receptor-positive
human breast cancer sections obtained from the Pathology Department of Sheba Medical
Center
• prpS6 - mouse pancreas
Scale bar = 100µm

Supplementary Table 1

Cy decreases the incidence of pregnancy in mice, and AS101 rescues it.

The fraction of females that became pregnant was calculated as the number of mice within each
group which conceived and delivered at least one litter throughout the 3 mating rounds.