Committing the Gonad to

Ovary Fate

Blanche Capel

Department of Cell Biology

Duke University Medical Center

The testis and ovary arise from the same primordia

E10.0-11.5

(mouse)

granulosa cells supporting cell lineage

steroidogenic cell lineage

primordial germ cell lineage spermatogonia

Leydig cells

Sertoli cells

Theca cells

oogonia

The bipotential gonad is composed of three bipotential lineages

Anne McLaren Paul Burgoyne Robin Lovell-Badge

The supporting cells are primed with an ovarian bias

Expression of the Y-linked Sry gene in the

supporting cell lineage drives testis fate

E10.0-11.5

(mouse)

E12.5

testis ovary

bipotential

Sry

The supporting cells are the first to show sexually dimorphic expression

a small number of transcripts are dimorphic in germ cells and all are X- or Y-linked

many genes have a pattern specific to supporting cell precursors that is shared between XX and XY cells

Sample global gene expression in gonads at 6 time points between E11.0-E12.0

Steve Munger

Sample gonads every 4 hours

Data from the time course revealed patterns of activation and repression in XX and XY gonads

XX Log Fold Change

XY Log Fold Change

Sox9

Dhh

Cbln1

XY Log Fold Change

Most male genes are activated in XY gonads

Anirudh Natarajan

XX Log Fold Change

Msx1

Wnt4

Irx3

Many female genes are repressed in XY gonads

XX Log Fold Change

XY Log Fold Change

Cascades of Dimorphic Expression –

Male-enriched genes

Fgf9

Acer3

Amh

Expression Trajectory

Cascades of Dimorphic Expression –

Female-enriched genes

Fst

Zfp277

Igfbp2

Expression Trajectory

XX XY

bipotential

?

But do adult

granulosa cells

directly stem

from this

population?

Adult Sertoli

cells develop

from supporting

cell precursors

Some female somatic cells can activate the Sry

promoter

GATA4

Sry-EGFP

Sry promoter

EGFP

The same cells also express FOXL2, a granulosa cell

marker

E13.5

Mork et al. Biol Reprod 2011

Sry-EGFP

FOXL2

Sertoli cells arise from the coelomic epithelium

during the bipotential period

After Karl et al., 1998

+ 2 h

+ 6 h

E11.75 +24 h E11.5 + 24 h

Mitotracker SOX9

Granulosa precursor cells arise from the XX

coelomic epithelium until E14.5

Labeled at E11.5-13.5

Mork et al. Biol Reprod 2011

trace 2

trace 3

birth

trace 1

trace 3

FOXL2+ cells are

mitotically arrested

from the time they

leave the surface

epithelium

E12.5

Mork et al. Biol Reprod 2011

Cyclins are specifically depleted in XX relative to XY supporting cells

FOXL2+ cells do not resume cycling until their

host follicle is activated

P1 P7

Ki67 FOXL2

p27 FOXL2

Follicle assembly

Do fetal FOXL2 cells form adult granulosa cells?

trace2

birth

sex determination

trace 1

Foxl2-

GFPCreER

R26R-lacZ

inject tamoxifen

Foxl2-GFPCreER; R26R-lacZ

Mork et al. Biol Reprod 2011

Fetal Foxl2-expressing-cells contribute granulosa cells to

medullary but very rarely cortical follicles

Two types of granulosa cells for two types of

follicles?

Cortical granulosa

cells

Medullary granulosa

cells

Cortical follicles are activated

during adult estrus cycles

Medullary follicles are

activated immediately after

follicle assembly at birth;

establish ovarian functionality

at the hormonal level prior to

the first LH surge

Follicle assembly

trace 2

trace 3

birth sex determination

Where do the cortical granulosa cells come from?

trace 1

BrdU Trace

Cortical granulosa cells arise from cycling

progenitors

Mork et al. Biol Reprod 2011

The ovarian surface epithelium gives rise to

pregranulosa cells between P1 and P7

Mork et al. Biol Reprod 2011

+ 24 h +72 h +96 h +96 h

P1

P3

P7

p27 DNA

Once specified, pregranulosa cells immediately

enter cell cycle arrest

Summary of mouse granulosa cell origins

Mork et al. Biol Reprod 2011

Ng et al., Nature Cell Biology, 2014 Rastetter et al, DevBio, 2014

Lgr5+ cells are present on the ovarian surface from E12.5 to adult stages, and act as progenitors for granulosa cells

Danielle Maatouk

• TSS/promoters • enhancers/repressors

• insulators

• LCRs

Isolate Supporting Cells for DNase Hypersensitivity Study

Sertoli-specific sites are identified by comparison to 6 other datasets

Enhancers of genes expressed in granulosa cells also have Sertoli-specific sites

Perhaps shared epigentic genome marks underlie

transdifferentiation of Sertoli cells into

granulosa-like cells in adult testes of Dmrt1 mutants

Matson et al. 2011

Transdifferentiation of granulosa cells into

Sertoli-like cells in adult ovaries

Uhlenhaut et al. 2009

Couse et al. 2003

SOX9

Adult induced Foxl2 KO

ERαβ KO

SOX9

THANKS Collaborators

Uwe Uhler – dcsb, Duke Greg Crawford – Duke

Steve Munger Samantha Jameson Anirudh Natarajan Danielle Maatouk Lindsey Mork Iordan Batchvarov Help from many others in the lab

NICHD, NIDDK

Duke Center for Systems Biology