and CYP3A mRNA and protein in different reproductive tissues of - - PowerPoint PPT Presentation

▶
and cyp3a mrna and protein in different
SMART_READER_LITE
LIVE PREVIEW

and CYP3A mRNA and protein in different reproductive tissues of - - PowerPoint PPT Presentation

Jan 07, 2023 624 likes •997 views

Expression study of ESR1, ESR2, CYP19 and CYP3A mRNA and protein in different reproductive tissues of breeding boars Asep Gunawan (1)(2) ., K.Kaewmala (2) .,U.Cinar (2) and K.Schellander (2) (2) Institute of Animal Science (1) Faculty of Animal

slide-1
SLIDE 1

Institute of Animal Science Animal Breeding and Husbandry group University of Bonn, Germany

Asep Gunawan(1)(2)., K.Kaewmala(2).,U.Cinar(2) and K.Schellander(2)

Expression study of ESR1, ESR2, CYP19 and CYP3A mRNA and protein in different reproductive tissues of breeding boars

(2)

Faculty of Animal Science Bogor Agriculture University

(1)

slide-2
SLIDE 2

2

Outline

  • Introduction
  • Boar Selection
  • Information of selected genes
  • Objective
  • Material and Methods
  • Work Flow of Expression Analysis
  • Results and Discussion
  • mRNA Expression Analysis
  • Protein Expression Analysis
  • Localization of Protein
  • Conclusion
  • Prospective aspect
slide-3
SLIDE 3

3

Introduction

slide-4
SLIDE 4

4

Boar Selection

AI has a significant influence on the pig breeding and production Fertility and sperm quality are important parameters for the selection of boars Difficult to perform direct selection: Low heritabilites Fertility (LS=0.01 – 0.06)1 Sperm quality (SC=0.14-0.18;SM=0.05-0.13)2 Effective way to improve male reproductive traits Marker Assisted Selection (MAS): candidate gene analysis

1See (2000); 2Brandt and Grandjot(2008)

slide-5
SLIDE 5

5

Boar reproductive physiology

slide-6
SLIDE 6

6

Reproductive tissues

Tissue Function Testes

  • Leydig cells produce testosterone under LH

stimulation

  • Sertoli cells produce estradiol under FSH stimulation

Caput

Early maturation in sperm

  • Translocation of cytoplasmic droplet
  • Change in membrane phospolipid

Corpus

Continued maturation of sperm

  • Change in membrane sterol:phospholipid
  • Increased permeability of plasma membrane

Cauda

Final maturation and storage of sperm

  • Increased intracelluler pH
  • Decrease intracelluler Ca2+
  • Eficient energy production
  • Improve pregnancy ensurance
  • Normal fertility and birth rate

Marengo et al (2008)

slide-7
SLIDE 7

7

  • Large number of genes and protein involved in the mechanism

and process of fertilization, but there a few report with an influence in sperm quality and fertility (Giesecke et al. 2009)

  • Fertility traits
  • Three genes ACTN1, ACR and ONPin6 had significantly effect
  • n boar fertility traits: non return rate and number of piglets

born alive (Wimmer et al.2005)

  • Sperm quality traits
  • Fourteen genes FSHB, PRL, ACR, INHA, INHBA, INHBB,

FST, RLN, RBP4, AR, ACTG2, GnRHR, OPNin6 and OPNpro significantly affected sperm quality traits: sperm concentration, semen volume per ejaculate, motility, plasma droplets rate and abnormal sperm rate (Lin et al. 2006a ; 2006b)

Literature studies

slide-8
SLIDE 8

8

Selected genes function in male reproductive trait

Gene Function Key tissue Reference ESR1

  • Association with semen traits : sperm number per

ejaculate and sperm motility;

  • Lack of either ESR1 result infertility in adult life
  • Spermatogenesis and sperm maturation

Testis Epididymis Terman et al., 2006; Guarducci et al.,2006; Eddy et al, 1996 Ren et al, 2008a Rohrer et al (1996) ESR2

  • Association genotype ESR2 gene to male infertility
  • Deficient ESR2 in male mice are reported to be fertile
  • Process of differentiation and maturation of testis

Testis, Epididymis Aschim et al (2006); Lambard et al (2004); Munoz et al (2002) CYP19

  • Catalyze

for estrogen synthesis from androgen; CYP19 deficiency caused progressive infertility in adult mice and reduced sperm production and sperm motility in humans Testis Furbass et al, 1997 Carani et al1997; Robertson et al, 1999; Herrmann et al, 2002, Tiwari et al 2008 CYP3A

  • Sperm maturity, sperm storage

Testis, Epididymis Ren et al, 2008

slide-9
SLIDE 9

9

Function of selected genes in reproductive tract

slide-10
SLIDE 10

10

Objective of the study

  • To study the mRNA expression and protein expression in testis

and epididymis between good and bad sperm quality of boar

  • To localize selected proteins in testis and epididymis
slide-11
SLIDE 11

11

Material and methods

slide-12
SLIDE 12

12

Sampling

4 good sperm quality 4 bad sperm quality AI station declaration

  • sperm concentration
  • sperm volume
  • sperm motility

Reproductive tissues

slide-13
SLIDE 13

13

Candidate gene selection from literature:

  • Association study
  • Gene expression

Semi-quantitative PCR

mRNA expression:

  • Semi quantitative PCR
  • RT-PCR

Protein expression:

  • Western blot
  • Immunofluorosence

Internal control : GAPDH (Glyceraldehyde 3-phosphate dehydrogenase)

  • Testis
  • Epididymis-head (caput)
  • Epididymis-body (corpus)
  • Epididymis-tail (cauda)
  • Brain
  • Bulbourethal gland
  • Testis
  • Vesicular gland
  • Epididymis-head - Prostate gland
  • Epididymis-body - Muscle
  • Epididymis-tail - Liver
  • Vasdeferent

Preliminary expression study: Reproductive and Non-reproductive Expression study: Good and bad sperm quality

ESR1 ESR2 CYP19 CYP3A

Workflow

slide-14
SLIDE 14

14

  • Gene expression analysis by SAS version 9.2, using the

Generalized Linear Model: Y = µ + a+ e Where Y : the expression of phenotype; µ : the overall mean a : the fix effect of phenotype (reproductive tissues:testis and epididymis good and bad sperm quality of boar) eij : the random residual error

Gene expression analysis

slide-15
SLIDE 15

15

  • Comparative mapping of selected genes ESR1, ESR2, CYP19 and

CYP3A were idetified using the INRA-Minnesota 7000 rad radiation hybrid panel (IMpRH) (Yerle et al. 1998)

  • Comparative maps analysis were performed using available sofwere

database https://wwwlgc.toulouse.inra.fr/pig/compare/compare.htm for chromosome assignment

Comparative mapping Sus scrofa <> Homo sapiens

slide-16
SLIDE 16

16

Results and Discussion

slide-17
SLIDE 17

17

The expression of selected genes in reproductive and non reproductive tissues

243 bp. GAPDH ESR1 CYP19 CYP3A 305 bp. 324 bp. 215 bp. ESR2 157 bp.

slide-18
SLIDE 18

18

The expression of selected genes in good and bad sperm qulity boars

GAPDH 243 bp. ESR1 CYP19 CYP3A 305 bp. 324 bp. 215 bp. ESR2 157 bp.

slide-19
SLIDE 19

19

The expression profile of ESR1 in good and bad sperm qulity boars

Testis

0.5 1 1.5 2 2.5 3

Good Bad

Relative Expression

Head of Epididymis

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6

Good Bad

Relative Expression Body of Epididymis

1 2 3 4 5 6

Good Bad

Relative Expression

Tail of Epididymis

1 2 3 4 5 6

Good Bad

Relative Expression

P<0.01

slide-20
SLIDE 20

20

Testis

7 8 9 10 11

Good Bad

Relative Expression

Head of Epididymis

10 11 12 13 14

Good Bad

Relative Expression

Body of Epididymis 9 10 11 12 13 Good Bad Relative Expression Tail of Epididymis 10 11 12 13 Good Bad Relative Expression

P<0.05 P<0.05

The expression profile of ESR2 in good and bad sperm qulity boars

slide-21
SLIDE 21

21

Testis 3 3.5 4 4.5 5 Good Bad Relative Expression

Head of Epididymis

7.5 8 8.5 9 9.5

Good Bad Relative Expression

Body of Epididymis

7.5 8 8.5 9 9.5 10

Good Bad

Relative Expression

Tail of Epididymis

6 6.5 7 7.5 8 8.5 9

Good Bad

Relative Expression

P<0.05

The expression profile of CYP19 in good and bad sperm qulity boars

slide-22
SLIDE 22

22

Testis

0.5 1 1.5 2 2.5 3 3.5

Good Bad

Relative Expression

Head of Epididymis

1 2 3 4 5

Good Bad

Relative Expression

Body of Epididymis 0.5 1 1.5 2 2.5 3 3.5 Good Bad Relative Expression Tail of Epididymis

1 2 3 4 5 6 7

Good Bad

Relative Expression

The expression profile of CYP3A in good and bad sperm qulity boars

slide-23
SLIDE 23

23

The expression protein of ESR1 and CYP19 in good and bad sperm quality boars

slide-24
SLIDE 24

24

Localization of ESR1and CYP19

FITC = Immunostain for Fluorescein Isothiocyanate (green) DAPI = Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (blue) NC = Negative Control

slide-25
SLIDE 25

25

Discussion

  • Up regulation of ESR1 was found in head of epididymis in boar with good

sperm quality → The highest concentration of estrogen receptor in the male reproductive tract is found in the head of epidydimis in mouse and macaques ( West et al,

1990: Shayu e al, 2005)

  • Down regulation of ESR2 was shown in testis and head of epididymis in

boar with good sperm quality → splicing of ESR2 gene might have specific function in spermatogenesis

(Forsti et al, 2003)

  • Up regulation of CYP19 was revealed in head of epididymis in boar with

good sperm quality → high level of CYP19 was detected in head of epididymis of rhesus monkey epididymis (Martinez et al. 2007)

1Janulis et al (1996); 2Forsti et al (2003); 3Martinez et al (2007); 4Malin et al (2000)

slide-26
SLIDE 26

26

Discussion

  • Localization of CYP19 was observed in leydig and epithelial cells

→The highest CYP19 activity and proliferation occured in the Leydig cells during puberty to adulthood (Hess et al, 2004)

  • ESR1 localization was detected in different part of epididymis

→ indicate important for regulating protein secretion and to be involved in the initiation of sperm motility (Pearl et al, 2007)

  • Localization of ESR1 was localized in the post acrosomal region

→ ESR1 was localized in post acrosomal region of the sperm head (Solakidi et

  • al. 2005)
  • ESR1was found to be localized on tail of sperm

→ involve in cell survival and motility (Aquila et al (2004)

1Hess et al (2004); 2Pearl et al (2007); 3Ramalho-Santos et al (2002); 4Aquila et al (2004)

slide-27
SLIDE 27

27

QTL for male reproduction trait

SSC 1 SSC 1

SPPJ TESTIWT TESTIWT

slide-28
SLIDE 28

28

ESR2 ESR2

QTL for male reproduction trait

AGEP AGEP AGEP

SSC 1

slide-29
SLIDE 29

29

QTL for male reproduction trait

SSC 1

NBA NBA NBA

slide-30
SLIDE 30

30

QTL for male reproduction trait

SSC 3 SSC 3

SEMVOL EPDW

slide-31
SLIDE 31

31

  • ESR1 gene mapped on SSC1p24-25

→ QTL for total sperm per ejaculate and close to the QTL for sperm, testicular weight (Xing et al, 2008; Ren et al, 2008)

  • ESR2 gene mapped on SSC1q22-27

→ QTL affecting reproductive traits for number of nipples and age at puberty (Cassedy et al, 2001)

  • The assignment of the porcine CYP19 gene mapped SSC1 q14-17

→ CYP19 to be a candidate gene affecting fertility performance in farm animals : bovine → 10q26; bufallo → 11q26 ; goat → 10q32.; sheep → chr 7 (Ianuzzi et al, 2001)

  • CYP3A gene mapped on SSC3, in the region p16-p17 or p11

→ QTL effect associated with semen volume and epididymal weight (Ren

et al, 2008)

Discussion

1Xing et al (2008); 2Ren et al (2008); 3Cassedy et al (2001); 4Ianuzzi et al (2001)

slide-32
SLIDE 32

32

Conclusion

  • We suggest that ESR1, ESR2 and CYP19 might be good candidate

genes for the sperm quality and fertility which could be used in boar selection

slide-33
SLIDE 33

33

Prospective aspect

Candidate gene study Association analysis Screening polymorphism Genotyping Sequencing analysis

SNPs analysis need to be done for ESR1,ESR2 and CYP19 as candidate genes for sperm quality and fertility traits

slide-34
SLIDE 34

34

Acknowledgement

  • DAAD
  • Prof Dr. Karl Schellander
  • Dr. Dawit Tesfaye, Dr. C. Phatsara, Mr. M. Ulas Cinar
  • Dr. Ralf Nolten
  • Prof. Dr. Mathias Becker, Mrs Susanne Hermes and Mrs Britta Chaffik
  • Ms. Kanokwan Kaewmala
  • Member of Animal Science
slide-35
SLIDE 35

35

Thank you for your attention!