Transcriptome changes during development in bovine ...

Transcriptome changes during development in bovine oocytes and early stage embryos Daniela IamarWno1, Francesco Strozzi1, Giovanna Lazzari2, Cesare Galli2 & John L. Williams1 1PTP – Parco Tecnologico Padano, Via Einstein, 26900 Lodi, Italy; 2AVANTEA- Laboratory of ReproducGve Technologies, Cremona, Italy

Corresponding author: [email protected]

AIM: To inves-gate stage-specific gene expression and the effect of genotype on the bovine embryo development and viability Introduction Fertilization and early cleavage stages are initiated by maternal RNA and proteins accumulated during oogenesis while large scale synthesis of messenger RNA from the diploid embryonic genome is initiated at the 8-cells stage in bovine.With knowledge of normal transcription patterns, it will be possible to identify errors in these early developmental events that are associated with embryonic losses. A global RNA gene expression analysis of bovine oocytes and early stage embryos was carried out using the bovine customArray Combimatrix 90K, designed with 43,768 unique probes, each representing a single bovine sequence. The effect of the genetic background of the embryo is being investigated to identify genes that are important for early development and viability.

Experiment 1

Experiment 2

²  transcriptomes of bovine MII oocytes and 8-cells, morulae and blastocyst stages of developing in vitro embryos

²  transcriptome of bovine MII oocytes recovered from large follicles of ovaries with pronounced follicular activity (Good) vs oocyte recovered from smaller and/or hypoplasic ovaries (Bad) to investigate the correlation between origin and quality of oocytes.

Oocyte and embryo collec-on:

Ø  5 pools of 15 Italian Holstein MII oocytes prepared from oocytes recovered from slaughterhouse and matured in vitro for 24 hours; Ø  5 pools of 8 cells (15), morulae (10) and blastocyst-stage (10) embryos were respecWvely collected 2, 5 and 7 days post-ferWlizaWon of oocytes from Italian Holstein cows ferWlized with Holstein semen

Oocyte collec-on

Ø  3 pools of GOOD vs 3 pools of BAD 15 Italian Holstein MII oocytes

MATERIALS AND METHODS

RNA purifica-on and amplifica-on • RNA extracWon was carried out using RNeasy Micro Plus isolaWon kit (Qiagen). • Total RNA quality was checked using RNA Pico 6000 kits on an Agilent 2100 Bioanalyzer. • (aRNA) was synthesised using the ampULSe amplificaWon and labelling kit (Kreatech biotechnology) from very small starWng samples by two successive rounds of in vitro T7 RNA transcripWon. • aRNA quality and concentraWon was determined using a Nano chip (Agilent Bioanalyzer) and A260/280 readings, respecWvely

aRNA Amer two rounds amplificaWon (250-1000 nt)

Cy5 Labelled aRNA

CombiMatrix CustomArray® 90K • The custom microarray was created using bovine transcript sequences downloaded from Ensembl release 50 and all the sequences from the bovine Unigene and dbEST databases (July 2008). • A pipeline wrijen in Perl was created to align the different sequences and select a unique set of minimally redundant bovine transcripts. • This dataset was used to design 43.768 unique probes of 35 nucleoWdes, each represenWng a single bovine sequence. • The probes were processed and synthesized in duplicate on a CombiMatrix CustomArray® 90K, along with negaWve and quality controls. • Data were analyzed using R and the Limma package from Bioconductor. • DifferenWally expressed genes were detected using Limma modelling analysis, with 0.05 as the Pvalue cut off.

Whole Expression Profiling

Scanning of CombiMatrix Array using GenePix 4000B scanner

RESULTS EXPERIMENT 1

In tables 1, 2, 3 and 4are shown the 20 top genes whose expression is activated between MII oocytes and blastocyst. The data show that several hundred genes were specific for each stage and a group of transiently expressed genes from MII Oocytes to 8-cells stage, at which the major activation of the embryonic genome occurs, and from 8-cells up to blastocyst, were investigate. Table 5 shows the genes under validation by qRT-PCR using SYBR-GREEN analysis. Among these, B-cell translocaWon 4 gene (BTG4) is a candidate maternal-effect gene found in maturing chicken oocytes(Elis et al, 2008); Zygote

arrest 1 like protein (Zar1L), is similar to Zar1 a known maternal-effect gene in the mouse (Mager et al, 2006); Bax Inhibitor 1 (BI-1), over-expressed in blastocyst, is an anW-apoptoWc protein and represents a new type of regulator of cell death pathways controlled by Bcl-2 and Bax; ID3 represent conserved negaWve regulators of human embryonic and induced pluripotent stem cell hematopoiesis (Seok-Ho Kong TABLE 5 - Specific-stage and transiently expressed genes under valida-on by qRT-PCR (in bold already validated genes) et al., 2010). Oocyte Oocyte to 8 cells

Table 1- Blastocyst vs MII Oocytes Annotation BTG4 ZAR1L ID-3 RGS2 SLC7A3 CPEB1 KRT18 PPIG PAIP1 TKT RGS16 BPGM FAM151A GNMT SPG21 BI1 AP4A MDHC PRR6 RNF187

log FC -8.05 -8.11 -7.37 -7.77 -7.06 -7.64 7.41 -7.12 -6.41 5.98 -6.61 -5.83 5.98 6.06 -4.16 4.75 -4.49 6.52 -6.32 4.38

Table 2- 8 cells vs MII Oocytes

Adj P-Value 2.71e-12 2.71e-12 4.03e-12 5.17e-12 6.70e-12 2.46e-11 2.64e-11 5.11e-11 6.19e-11 8.62e-11 1.56e-10 2.44e-10 2.44e-10 2.65e-10 2.79e-10 2.91e-10 2.91e-10 3.34e-10 3.49e-10 3.51e-10

Annotation TRIM4 JAZF1 gamma-taxilin FANK1 USH2A CCDC92 CASP14 VATE1 GLRX3 Fructose-bisphosphate aldolase Intraflagellar transport protein 20 homolog RPA2 family Malate dehydrogen., mitochondrial precursor U11/U12 small nuclear ribonucleoprotein Enoyl-CoA hydratase, mitochondrial precursor Zinc finger protein 323 Mitoch import inner membrane transl subTim9 RPA2 family Retrotransposed gene Ubiquitin-conjugating enzyme E2 E1

RESULTS EXPERIMENT 2

Table 3- Morulae vs MII Oocytes logFC 3,97 3,52 -2,80 3,71 4,30 2,05 2,18 3,74 4,35 3,29 3,01 2,74 2,86 3,41 2,54 2,96 3,41 2,69 3,16 2,15

adj.P.Val 1,53E-10 1,70E-10 2,61E-10 2,61E-10 3,62E-10 7,61E-10 1,11E-09 1,35E-09 1,36E-09 1,36E-09 1,36E-09 1,82E-09 1,82E-09 1,86E-09 1,86E-09 1,87E-09 1,87E-09 2,96E-09 4,63E-09 5,20E-09

Annota-on BTG4 TKT A0JNF2 Q3SZI3_BOVIN ZAR1Like CENPV STARD7 S10AE_BOVIN GNMT PMGE TRA2B_BOVIN CYTX or Stefin-C FOLR1 FerriWn like IDHP_BOVIN CATL1_BOVIN ID3_BOVIN WDR55_BOVIN MPCP_BOVIN RPAC1_BOVIN

logFC -7.39 7.47 -6.56 4.87 -6.12 -6.42 5.71 6.42 6.58 -5.99 4.92 5.42 6.22 -7.34 5.02 5.66 -6.26 5.64 4.52 6.31

Table 4- 8 cells vs Blastocyst adj.P.Val 3.68e-24 8.65e-20 1.55e-19 1.55e-19 3.34e-19 3.51e-19 5.17e-19 8.39e-19 4.96e-18 1.26e-17 1.64e-17 1.66e-17 2.05e-17 2.05-17 4.21e-17 4.31e-17 5.84e-17 6.23e-17 6.54e-17 1.03e-16

Annotation

logFC

adj.P.Val

ZAR1-LIKE

7,77

2,10E-21

ID-3

7,21

2,10E-21

BTG4

7,76

2,10E-21

RGS2

7,53

6,95E-21

KRT18

-7,55

4,89E-18

SLC7A3

6,56

1,79E-17

BI-1

-5,20

2,14E-17

BPGM

5,95

2,18E-17

SLC25A3

-6,23

2,50E-17

PRR6

6,33

4,86E-17

FAM151A

-5,74

5,37E-17

Protein S100-A14

-7,55

5,62E-17

Maspardin

4,17

9,61E-17

START domain containing 7

-5,50

1,73E-16

Cathepsin L1

-7,13

2,44E-16

Transketolase

-5,25

2,60E-16

pantothenate kinase 3

-4,64

2,97E-16

RAD17

5,00

2,97E-16

GDF-9

6,87

4,08E-16

CKS-1

3,42

4,83E-16



from 8 cells to blastocyst

Morulae

Elis S. et al, BMC Genomics 2008, Feb 29 9:110; Galli C. Et al, 2006, proceeding of Congresso Annuale della Società Italiana per il progresso della Zootecnia: 77-86; Fair T. Et al, Theriogenology 2007: S91-S97; Mager J. et al, Mamm Genome 2006, 17: 941-949; Niemann H. Et al, Theriogenology 2007 S165-S177; Seok-Ho Kong et al, Journ. Of Cell Science 2010: 124, 1445-1452; Vigneault C. Et al, ReproducWon Res. 2009; 137: 245-257.

Morulae-Blastocyst Blastocyst

U1SBP ZNF323

IDPH GLRX3

RBMX MBD1

TKT WDR55

KRT18 KRT8

ID3 PMGE FerriGnLike RGS16 SPG21 SBP1 CENPV

H4 JAZF1 FANK1

V-ATPase subunit 1

ING1 COHA1_Bovin

SH3BGRL3 PLAC8









CATL1 MPCP POLR1C ALKBH4 RNF187 TRA2B START S10AE UDPglu AP2gamma CytX FOLR1 TROPα1 NANOG BI1 FAM151A RAB14

CONCLUSIONS

This experiment to observe differences between MII oocyte recovered from slaughtered ovaries with pronounced follicular activity vs oocyte recovered from smaller and/or hypoplasic ovaries did not give significant differentially expressed genes. 1 References

MEST BTG4 SQSTM1 ZAR1Like

8 cells



These results show that the RNA preparation protocol and the bovine CombiMatrix CustomArray® 90K can be used successfully for global gene expression analysis in bovine oocytes and embryos, to understand key mechanisms relevant to embryo development. The characterization of and embryonic transcriptomes is essential for investigating genes involved in early development and potentially for improving assisted reproductive techniques. With knowledge of normal transcription patterns, it will be possible to identify errors in these early developmental events that are associated with embryonic losses.

3rd EmbryoGenomics Mee4ng , September 20th-22nd, 2011 – Bonn, Germany

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