Originally published 25 February 2016; corrected 31 March 2016

www.sciencemag.org/cgi/content/full/science.aad2156/DC1

Supplementary Materials for

Mouse oocytes differentiate through organelle enrichment from sister

cyst germ cells

Lei Lei* and Allan C. Spradling*

*Corresponding author. E-mail: spradling@ciwemb.edu (A.C.S.); leile@med.umich.edu (L.L.)

Published 25 February 2016 on Science First Release

DOI: 10.1126/science.aad2156

This PDF file includes:

Materials and Methods

Figs. S1 to S6

Tables S1 and S2

Full Reference List

Correction: Table S2 was inadvertently omitted from the previous version. It has been

added here. Also, a statement about the frequency analysis of germ cells has been

added to the section on statistics.

SUPPLEMENTARY MATERIAL

Supplementary Materials and Methods

Materials

Animals

CAG-CreER mice (004682) and R26R EYFP mice (006148) were acquired from the

Jackson Laboratory. All mice were genotyped according the protocols from the JAX®

Mice database.

Antibodies

Antibodies used are summarized in Table S1.

Methods

Single-cell lineage labeling

Tamoxifen-induced single primordial germ cell (PGC) lineage labeling was performed as

described (9). To obtain fetuses with lineage-labeled germ cells, adult female R26R-

EYFP mice were mated with male CAG-creER mice. 12 PM on the day a vaginal plug

appeared was designated as embryonic day 0.5 (E 0.5). Birth usually occurred between

E19 and E20. The day of the birth designated as postnatal day 0 (P0). Pregnant female

R26R-EYFP mice at E10.5 received a single intraperitoneal injection of Tamoxifen

(Sigma), 0.2 mg per 40g body weight, to label on average one PGC per ovary. All

procedures were approved by IACUC.

Whole-mount immuno-staining of mouse fetal ovaries

Fetal ovaries were dissected in cold phosphate buffered saline (PBS), and fixed

immediately in cold 4% paraformaldehyde (PFA) for 2-5 hours. After washing in PBST2

(PBS with 0.1% Tween-20 and 0.5% Triton X-100) for 1 hour, fetal ovaries were then

incubated with primary antibodies overnight. After secondary antibody incubation on the

next day, ovaries were washed in PBST2 and stained with DAPI to visualize cell nuclei.

Ovaries were analyzed using confocal microscopy (Leica SP5) and stacks of serial

images of each germ cell clone were acquired. Three-dimensional models were

generated using Imaris software (Bitplane).

Single-cell apoptosis assay

Ovaries from P0 fetuses were dissected and digested in 500 μl PBS with 0.25% trypsin

and 2.5 mg/ml colleganase at room temperature. Disassociated tissue was centrifuged at

500 g for 5 minutes to collect cells. Cells were then resuspended and washed with PBS.

Cells were centrifuged again and resuspended with 500 μl DMEM-F12 media with 3

mg/ml bovine serum albumin (BSA). Annexin-V antibody was added into the media

with cells at a dilution of 1:10. A poly-L-lysine coated cover slip (BD biosciences) was

then placed into one well of a 4-well cell culture plate. The cell suspention was added on

the cover slip and the cells were cultured for one hour. Afterwards, the cell culture media

was removed and the cover slip with adherent cells was washed with PBS beriefly and

fixed using 4% PFA for 30 minutes. After fixation, cells were washed with PBST2 and

incubated with GM130 antibody to stain Golgi and MVH (VASA) antibody to stain germ

cells.

Fetal ovarian culture

Mouse fetal ovaries were dissected on E17.5 and cultured in 500 μl basal cell culture

media: DMEM-F12 media supplemented with 3 mg/ml bovine serum albumin (BSA).

Each culture contained three ovaries on a membrane insert (Millipore) in one well of a

24-well cell culture plate. After incubation for two days with or without inhibitor, the

ovaries were removed, washed with basal media three times, and then placed onto a new

membrane with fresh basal media and incubation was continued. After 6 total days in

culture, corresponding to postnatal day 4 in vivo, ovaries were fixed in 4% PFA for

further analysis. The inhibitors used were Ciliobrevin D (25 μM) and Colchicine (10

nM).

Quantifying germ cell number

The determine the number of germ cells per ovary, fetal ovaries cultured as described

above were fixed in 4% PFA at 4 degree for 3 hours. After washing in PBS for 10

minutes, the ovaries were placed in 30% sucrose at 4 degree overnight. On the next day,

the ovaries were embeded in optimal cutting temperature medium (OCT) and sectioned at

10 μm. Serial sections of a whole ovary were collected and stained with MVH antibody

to stain germ cells. To calculate the total germ cell number in each ovary, every 5th

section was counted throughout the entire ovary, beginning with the first section, and the

final count was multiplied by 5 as a correction factor (19, 20).

Centrosome quantification

Centrosomes were stained with anti-pericentrin and anti-γ-tubulin antibodies. The amount

of pericentriolar material within clusters in a germ cell was measured using serial

confocal images of each pericentrin-immunostaining cluster by using Image J. The x, y

diameter of the biggest optical cross section and the z diameter of each pericentrin cluster

were measured to calculate three radii (Rx, Ry and Rz). The volume of a centrosome

cluster (Vcen) was then calculated using Vcen =4/3*3.14* Rx* Ry*Rz. For germ cells with

multiple pericentrin-positive clusters, only foci the size of clusters in E14.5 germ cells or

larger were measured and sumed to get the total volumes of pericentriolar material in the

cell. The amount of centrosomal material in each germ cell was also quantified based on

γ-tubulin staining using the same method. All γ-tubulin positive foci in the germ cell were

measured and summed to get the total volume. About 100 germ cells at each stage were

measured.

Golgi quantification:

The approximate surface area of Golgi membranes in each germ cell was quantified as

follows. Serial confocal images of GM130 immunostaining were analyzed using Image

J. Golgi were modeled as ellipsoids, hence the x and y diameters of the biggest cross

section and the z diameter of each Golgi segment were measured, and the x,y, and z radii

calculated by diving the diameters by 2. The surface area was then calculated by using

the equation:

Sgol= 4*3.14*(((Rx*Ry)^1.6+(Rx*Rz)^1.6+(Ry*Rz)^1.6)/3)^0.63

For germ cells in P0 and P4 ovaries, only germ cells with a large Golgi sphere (Balbiani

body) were analyzed. About 100 germ cells at each stage were measured.

Mitochondial quantification:

The number of mitochondria per germ cell at different ages was counted and calculated

using EM images. Germ cell sections of near maximal cross section based on nuclear

diameter were selected and the number of mitochondria counted (Nmit s). Germ cells

whose nuclear diameters in section were larger than 8 μm at E14.5, and 10 μm at E17.5

were analyzed. For germ cells at P0 and P4, only cells with a Balbiani body were

counted. The diameter of about 100 mitochondira were also measured at each stage to

calculate the average (Dave mit ). The volume of the germ cells stained with VASA at each

stage was calculated based on confocal images. The diameter of 100 germ cells was

measured to calculate the average radius of germ cells (Rave gc), the volume of germ cells

(Vgc), Vgc=4/3*3.14*Rave gc^3 and the surface area of the largest cross section (Slgc),

Slgc=3.14* Rave gc^2. For germ cells in P0 and P4 ovaries, only cells with a Balbiani body

(Golgi sphere by GM130 staining) were measured. The number of total mitochondia in a

germ cell (Nmit total) was then calculated by using the equation: Nmit total = Vgc /(Slgc* Dave mit)*

Nmit s. About 100 germ cells at each stage were measured.

Statistics:

Data are presented as mean±SD. One-way ANOVA was used to analyze the difference

between to experimental groups. A level of P< 0.05 was considered to be statistically

sigificant. For the frequency analysis of germ cells with > 3 bridges in Fig. 1F, only

cysts with at least 4 cells were considered, to mitigate the effects of apoptosis and bridge

loss.

Supplementary Tables

Table S1. Antibodies

Antibody Vendor Dilution

AnnexinV Santa Cruz (SC-1929) 1:100

AIF Cell Signaling (#4642) 1:100

Fibrillarin Cell Singaling (#2639) 1:100

Gamma-tubulin Abcam (ab179503) 1:100

GFP Aves Labs (GFP-1020) 1:1000

GM130 BD biosciences (610822) 1:100

HP1beta Abcam (ab10811) 1:100

MVH Abcam (ab13840)

Pericentrin Dr. Yixian Zheng (Carnegie

Institution for Science)

1:1000

Phalloidin Life Technology 1:500

Tex14 Abcam (ab41733) 1:100

Table S2. Cyst structures determined by reconstruction.

Cyst totalcells 4bcells 3bcells 2bcells 1bcells 0bcellsE14.51 12 0 2 5 4 1

2 14 1 2 6 5 03 14 0 2 7 5 04 15 0 3 5 7 05 18 1 4 3 8 26 251 1 10 13 07 30 0 7 9 12 28 8 1 1 0 5 19 8 0 0 5 2 1

10 2 0 0 0 2 011 22 0 3 9 8 212 16 0 1 6 8 113 16 0 0 6 5 514 5 0 0 3 2 015 30 1 2 13 11 216 6 0 1 4 1 017 12 0 3 3 4 218 14 0 2 7 5 019 4 0 0 2 2 020 4 0 0 2 2 021 5 0 0 1 4 022 7 0 0 3 3 123 8 0 3 0 5 0

E17.5*1 2

0 2 12

3

3 6

1 4 2

0

5 2

0 6 2

0

7 3

0 8 11

3

9 3

1

10 7

3 11 3

0

12 3

1 13 5

2

14 2

0 15 2

0

16 13

4 17 7

1

18 2

0 19 2

0

20 2

1 21 3

0

22 4

1 23 6

1

24 2

0 25 2

0

26 15

2 27 4

1

28 8

1 29 8

1

30 5

1 31 2

0

32 7

2 33 9

3

34 25

3 E18.51 4 0 0 4 0 0

2 2 0 0 0 2 03 2 0 0 0 2 04 2 0 0 0 2 05 2 0 0 1 1 06 3 1 0 0 2 07 3 0 1 1 1 08 6 0 0 4 2 09 5 0 1 2 2 0

10 6 2 1 1 2 0Lineage-labeled ovaries of the indicated age were stained with Tex14 and the

interconnection patterns of cysts were determined following modeling using Imaris

software *E17.5 data are presented as “number of the germ cells with ≥ 3 bridges”

Supplementary Figure Legends

Figure S1. Tex14 is not found on ring canals prior to E14.5. A representative mouse

ovarian cyst is shown at E12.5 stained for lineage (YFP, green) and Tex14 (pink). Small

aggregates (arrows), but no ring structures are seen until the end of cyst formation at

E14.5.

Figure S2. Putative transferred centrosome(s) are morphologically distinct from the

bipartite meiotic centrosome. Arrowhead indicates meiotic centrosomes, which remain

distinct form a putative transferred centrosome (arrow) in a lineage-labeled (red) E18.5

germ cell. The germ cell in the lower right shows the same two types of centrosome.

Pericentrin (green).

Figure S3. Transfer of nurse cell cytoplasm and Balbiani body formation. (A, B) B-

body containing cells preferentially lack expression of the apoptosis marker AnnexinV

(p<0.001; Fisher’s exact test). (C) B-body containing cells are selectively preserved. (D)

Electron micrographs of an E17.5 (upper) and a P0 (lower) fetal germ cell in which the

cytoplasm and nuclei appear to be separating.

Figure S4. In vitro ovarian culture recapitulates normal development. (A,B) The

number of germ cells per ovary in normal ovaries in vivo (A) is about the same as in

E17.5 ovaries cultured in vivo for the indicated times (B). (C) The average germ cell

volume of E17.5 cells, and subsequently of Balbinai-body containing cells, following

culture for indicated times, resemble the volumes of germ cells developing for

corresponding times in vivo. D) The amount of Pericentrin per cell of E17.5 cells, and

subsequently of Balbinai-body containing cells, following culture for indicated times,

resemble the amount in germ cells developing for corresponding times in vivo.

Figure S5. Analysis of microtubule-dependent oocyte differentiation in vitro.� (A)

Microtubule inhibitor treatment does not significantly impact the rate of somatic cell

division in vitro. Percentage of KI67 positive somatic cells in cultured mouse fetal

ovaries after a two-day inhibitor treatment (E17.5-E19.5). �(B) Treating ovaries with

microtubule inhibitors beginning at E19.5 causes a small but significant reduction in

Balbiani body formation. Percentage of germ cells with a Balbiani body in mouse fetal

ovaries after 6-day culture (from E17.5) with the inhibitor treatment from E19.5(P0) to

P2.� (C) During prolonged culture in vitro without inhibitors, many follicles begin to

grow. Growth is blocked by microtubule inhibitors. Histological sections of the ovaries

after 9-day culture (from E17.5). (D-E) Quatification of the effect of inhibitors on germ

cell number and growth in fetal ovaries cultured for 9 days (from E17.5). (F) Histogram

showing the total oocyte volume and Balbiani body volume in wild type oocytes at P4.

Considerable heterogeneity is observed.

Figure S6. Summary model of mouse oocyte differentiation based on organelle and

cytoplasm transport from sister germ cells within germline cysts. Mouse oocyte

differentiation initiates at E10.5, when primordial germ cells (PGCs) start incomplete

cytokinesis to form germline cysts. Germ cells in cysts are connected by intercellular

bridges. Around E17.5, organelles in cyst germ cells reorganize and transport through the

intercellular connections into the germ cells with a forming Balbiani body (curved

magenta). Note bridges are found dissociated from the cell connections during large scale

organelle (centrosomes in green; Golgi in magenta; mitochondira in blue) and cytoplasm

transport. Germ cells with a Balbiani body (magenta circle), which is the highly orgnized

organlle cluster, differentiate into primary oocytes with enriched cytoplasm. Germ cells

that donate organelles undergo apopotsis mostly from P0 to P4.

Fig.  S1  

Fig. S1

Fig.  S2  

Fig.  S3  

Fig. S3

Fig.  S4  

Fig.  S5  

Fig.  S6  

Fig. S6

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