Gene expression on oogenesis and oocyte development 2 Gene... · Gene expression on oogenesis and...

Ovarian Club VII

Gene expression on oogenesis and

oocyte development

Peking University Third Hospital

Qiao Jie

2016-05

Fertility is declining

WHO human reproductive special programme report:

The infertility rate is as high as 15%-20% in the

world,becoming the third largest disease after cancer and

cardiovascular disease abroad

In the world 60-80 million couples

In China 12-15 million couples

1

Affected their normal work and living

Lead to broken families, unharmonious society

The decline in the quality of the birth population

Oocyte quality！！！

Gene expression and regulation！！！

Oogenesis and oocyte development

2 http://buffonescience9.wikispaces.com/UNIT+3+-+Cell+Reproduction

Stages of oogenesis and oocyte development

3 Jessica Azoury et al. Biol Cell, 2009

Griffin J et al. JECAR, 2006

Follicle size Oocyte size Granulosa cell growth and follice size

人人人

人

人

人

human

mice

鼠鼠鼠

鼠

鼠

鼠

The species difference in oocyte development

4 McGee EA et al. Endocr Rev, 2000

http://edrv.endojournals.org/content/21/2/200/F2.large.jpg



mouse

2 human

1

5

Gene expression on oogenesis and oocyte development

Telfer et al. Fertil Steril, 2013

http://csls-text.c.u-tokyo.ac.jp/active/12_05.html

透明带


6

Oogenesis in mammals involves a complex series of morphogenetic changes

that occur within the ovarian follicle. Many genes involved in the mouse

oogenesis and oocyte development

The outline of PGC specification

1、PGC specification

2、PGC migration

3、colonization in the gonads

7 Mitinori Saitou et al. Cold Spring Harb Perspect Biol, 2012

Genes specific to mouse PGC

8

11 genes were identified

3 genes are novel

Specifically expressed

in M and F fetal germ cells

both in vivo and in vitro

Not expressed in ESCs

The identified genes have homologs in humans demonstrating their evolutionary

conservation indicating their functional importance. By using these genes as germ cell

markers, garnering significant insight into human reproduction and fertility were expect

Tripartite transcriptional network for PGCs

9

BLIMP1, AP2γ and PRDM14 are

sufficient for PGC specification

透明带

10




RSPO1/β-Catenin signaling pathway regulates oogonia

differentiation

11

透明带

12




Gene expression during folliculogenesis

13

Several genes were also found to be differentially express pattern, including

members of the growth factor family, such as c-kit, fgf, lif, kgf, bmp-4 , cell-cycle

regulators and hormones, extra-cellular matrix genes, and cytoskeletal genes ,

transcriptional regulators and signal transducers, immune response genes.

Selective degradation of transcripts during GV to MII

transition

14

Transcriptome analysis of MII oocyte

15

16

Proteome of mouse oocytes at different developmental stages

The analysis of GV oocytes, MII oocytes, zygotes proteomics revealed the

differences of protein expression in individual stages.

Regulation genes in oogenesis and oocyte development

Jagarlamudi K et al. Mol Cell Endocrinol., 2012

17

Richards JS. et al. J Clin Invest, 2010

18

Regulation genes in oogenesis and oocyte development

Some proteins involved in mouse oocyte meiosis regulation

19

The effect of Non-coding RNA on mouse oocyte development

20

Ago2 is a protein

which is important

for siRNAs trigger

endonucleolytic

cleavage of target

mRNAs

21

Non-coding RNA play an important role during

oocyte development

Veselovska L et al. Genome Biol, 2015

Granulosa cells play crucial roles during mouse

oocyte development

Meijia Zhang et al. Science

2010;330:366-369 Heng-Yu Fan et al. Science

2009;324:938-941

Oocyte maturation was

blocked after ERK1/2

depletion in granulosa cells

Oocyte could not sustain GV stage after

NPPC or its receptor NPR2 depletion which

were expressed in mural granulosa cells and

cumulus cells, respectively

22

The damage of acrylamide on mouse oocyte

quality

High fat diet affect mouse oocyte

quality

The impact of environment and high fat diet on oocyte quality

23

mouse

2 human

1

24


透明带

25




Transcriptome of human PGCs from the migrating

stage to the gonadal stage

Both pluripotency genes and germline-specific genes are expressed in

human PGCs

26

Gene expression of 16–16.5 weeks from fetal testes, fetal ovaries and hESCs

PGC

The ontogeny of cKIT+ human primordial germ cells: A resource

for human germ line reprogramming, imprint erasure and in vitro

differentiation

Sofia Gkountela1,2, Ziwei Li1,2, John J. Vincent1,2,3, Kelvin X. Zhang4, Angela Chen5, Matteo

Pellegrini1, and Amander T. Clark1,2,3,6

1Department of Molecular Cell and Developmental Biology, University of California Los Angeles,

Los Angeles, California, 90095; United States of America

2Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of

California Los Angeles, Los Angeles, California, 90095; United States of America

3Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, 90095;

United States of America

4Department of Biological Chemistry, Howard Hughes Medical Institute, University of California

Los Angeles, Los Angeles, California, 90095; United States of America

5Obstetrics & Gynecology, David Geffen School of Medicine, University of California Los Angeles,

Los Angeles, California, 90095; United States of America

6Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles,

California, 90095; United States of America

Abstract

Generation of research quality, clinically relevant cell types in vitro from human pluripotent stem

cells (hPSCs) requires detailed understanding of the equivalent human cell types. Here we

analyzed 134 human embryonic and fetal samples from 6–20 developmental weeks and identified

the stages in which cKIT+ primordial germ cells (PGCs), the precursors of gametes, undergo

whole genome epigenetic reprogramming with global depletion of 5mC, H3K27me3, H2A.Z and

the time where imprint erasure is initiated and 5hmC is present. Using five alternate in vitro

differentiation strategies combined with single-cell microfluidic analysis and a bona fide human

cKIT+ PGC signature, we show the stage of cKIT+ PGC formation in the first 16 days of

differentiation. Taken together, our study creates a resource of human germ line ontogeny that is

essential for future studies aimed at in vitro differentiation and unveiling mechanisms necessary to

pass human DNA from one generation to the next.

The foundation of human health at a cellular and molecular level is built upon accurate

lineage differentiation during embryonic and fetal life. In recent years, a major barrier to

study human development was overcome through the generation of human pluripotent stem

Correspondence should be addressed to: A.T.C. ([email protected]).

AUTHOR CONTRIBUTIONS

S.G. designed and performed the experiments, analyzed data and wrote the manuscript; Z.L. performed flow analyses, RNA and DNA

extraction for some gonadal samples, J.J.V. performed the single cell analysis of female ovary at 14 weeks; K.X.Z. and M.P.

performed the RNA-Sequencing data analysis; A.C. provided gonadal samples used in this study; A.T.C. designed the experiments,

analyzed data and wrote the manuscript.

COMPETING FINANCIAL INTERESTS

The authors declare no conflict of interest.

NIH Public AccessAuthor ManuscriptNat Cell Biol. Author manuscript; available in PMC 2013 September 30.

Published in final edited form as:

Nat Cell Biol. 2013 January ; 15(1): 113–122. doi:10.1038/ncb2638.

NIH

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Fig. 5.

RNA-Seq reveals the transcriptional identity of cKIT + PGCs. Single cell analysis of hESCs

shows stochastic expression of germ line genes. ( a) Heat map of 5,455 differentially

expressed genes (p value < 0.05) in at least one of three comparisons (male cKIT + vs H1

hESCs; female cKIT+ vs H1 hESCs; male cKIT+ vs. female cKIT+). The enriched GO terms

in the 13 resulting clusters are shown. ( b) Heat map of Pearson Correlation Coefficient

scores between hESCs and cKIT+ male and female PGCs. (c) Heat map of FKPM values for

selected genes in hESCs and cKIT+ male and female PGCs. Abbreviations: M= Male, F=

Female. (d,e) Heat map of GAPDH (G), OCT4 (O), BLIMP1 (B), DAZL (D), VASA (V),

NANOS3 (N3) and NANOS2 (N2) for H1 hESCs in triplicate (columns) in 100, 10, 0 or

single TRA-1-60+ cells (rows) sorted from H1 (d) and UCLA1 (e) hESCs. (f) Heat map as

in d and e plus cKIT (K) for TRA-1-81+/cKIT+ H1 hESCs. (g) Gating strategy to sort

TRA-1-81+/cKIT+ cells from the H1 hESC line. cKIT+ cells are gated from the TRA-1-81+

fraction, using a FITC secondary antibody against side scatter (SSC).

Gkountela et al. Page 15

Nat Cell Biol. Author manuscript; available in PMC 2013 September 30.

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透明带

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Ovarian

tissue

Enzyme digestion

5 group of follicle Affymetrics array detection

of each group

RNA extraction

Kristensen SG et al. Mol Cell Endocrinol, 2015

Expression profile microarray of follicle

29

RNA microarray analysis for follicle or granular cell

gene expression profiles

30

Transcriptome analysis of MII oocyte

31

PGCs Primordial

follicles

Primary Preantral Early antral Pre-ovulatory

activate

meiosis

PGCs fomation

Depend on

ovary

Non-gonadotropin-dependent Gonadotropin-dependent

Sánchez F et al. Biochim Biophys Acta, 2012

Genes in human oogenesis and oocyte development

32

•The follicles are activated in PTEN

knock-out mice.

•In PTEN inhibitor activated neonatal

mouse ovary，primodial follicles were

activated. After transplantation

mature oocytes were fertilized in vitro

and progeny mice were obtained

•PTEN inhibitor Akt activated follices

in POI patient and live birth was

obtained.

Li J, et al. PNAS, 2010

Deepak A et al. PLoS ONE, 2012

Kazuhiro K, et al. PNAS, 2013

The activation of primodial follicle：PTEN inhibitor

33

In 2016, studies on NEJM indicated mutations in TUBB8 was

associated with human oocyte meiotic arrest 34

TUBB8 in human and mouse oocytes cause abnormal

spindles and maturation defects

36

37

For the first time Chinese scholars found the "coat"

of human oocyte and its pathogenic genes

Yu et al. Cell, 2013

Our group establish the first human female personal genetic map

38

Hou*,Fan*,et al,Cell,2013

Whole genome amplification (WGA),

sequencing and haplotype analysis of single

human oocytes, polar bodies and blastocyst

cells, based on MALBAC technique

The sequencing method for the genome

of polar body has been proposed.

Lysis WGA + Target Enrichment

Low depth NGS

Blastocyst biopsy

A T

C G

A T

C

G

G C

C G T

A

T A G

C

A T

C G T

A

G C

C

G

C G T

A G C

T A

G C

C G C

G T A

CNVs for aneuploidy detection

SNVs for monogenic disease diagnosis

C A C G A A C C T T

Linkage analysis

Cell lysate WGA product with

targeted site enrichment

Genome sequences

T A G

G A C

Mutation site A C

Yan L…..Qiao J. Proc Natl Acad Sci U S A. 2015.

112(52):15964-9.

Simultaneous avoidance of monogenic disease and chromosome

abnormality by next-generation sequencing with linkage analyses

Mutated allele revealed by sequencing with aneuploidy and linkage analyses

(MARSALA)

2014.11.30 2014.9.19

Wife carries the disease-allele

X-linked hypohidrotic ectodermal

dysplasia

Husband carries the

disease-allele

Hereditary multiple exostose

Both of the couple carries

the disease-allele

Spinal Muscular Atrophy

Completion of PGD/PGS for 27 cases with 24 kinds of

monogenic disorders.

Seven cases have received embryo transfer, and 5 of them

achieved successful pregnancy.

The diagnostic accuracy rate is 100%.

Translational Medicine

Clinical Application of new methods in PGD/PGS

2015.12.4 DOB:

• Isolation procedure

• Novel biomaterial

• Certain growth factors

In vitro follicle culture

39

Review of human follicle culture in vitro

Development potential of follicle in vitro is limited

No. Origin of

ovary

Stage

started

Day of

culture

Culture

system

Results Authors

1 Adult Preantral 120h 2D P increase Roy SK. Fertil Steril. 1993

2 Adult Preantral/E

arly antral

2 weeks 2D E2 increase Abir R. Fertil Steril.1997

3 Adult

Primary/Sec

ondary

24h 3D GCs

proliferation

Abir R. Hum Reprod.1999

4 Adult

Preantral/E

arly antral

4 days 2D Antral formation Evelyn E. Hum Reprod. 2008

5 Adult

Preantral/E

arly antral

30 days 3D Antral formation Min Xu. Hum Reprod. 2009

6 Adult

Preantral 7 days 3D Diameter

increased

Christiani A. Hum Reprod. 2009

7 Adult

Preantral 7 days 3D Diameter

increased

J Vanacker. Fertil Steril. 2011

8 Adult

Primary/Sec

ondary

7 days 3D Diameter

increased

J Vanacker. Fertil Steril. 2013

9 Child/Adult Secondary 6 days 2D Diameter

increased

R. Anderson. Hum Reprod.2014

40

Engineering the ovarian cycle using in vitro follicle culture model

41

Human follicle culture in vitro：the effect of bFGF

Day 0 Day 2 Day 4 Day 8

The diameter and survival rate of the follicles and the

percentage with good viability are higher in bFGF

treated group.

42 Wang TR et al. Hum Reprod, 2014

Mesechymal stem cell (MSC) co-culture with human pre-

antral follicle promoted the viability of follicles

43

MSC promoted the viability of follices in vitro

Xia X et al. Reprod Sci, 2015

Improve maturation system in vitro—add growth factors

Effects of combined epidermal growth factor, brain-derived neurotrophic factor and

insulin-like growth factor-1 on human oocyte maturation

EGF, BDNF and IGF-1 can improve oocyte maturation rate and quality in vitro, and

consequently increase early embryo development and blastocyst formation

44 Yu Y et al. Hum Reprod, 2012

45


Richards JS. et al. J Clin Invest, 2010; Sánchez F et al. Biochim Biophys Acta, 2012

Thank you for attention！

Gene expression on oogenesis and oocyte development 2 Gene... · Gene expression on oogenesis and...

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