Genetic factor in asthenozoospermic patient: relevance to laboratorium test

and clinical management

Asmarinah

UNIVERSITY OF INDONESIAFACULTY OF MEDICINEDEPARTMENT OF MEDICAL BIOLOGY

Introduction

Cause of male infertility

Low sperm motility (Asthenozoospermia)

< 50 % sperm with progressive motility (category a + b)*, or< 25 % sperm with rapid and progressive motility (category a)*

* Category of sperm motility:a. Rapid and progressive motility (> 25 um/sec)b. Slowly and progressive motilityc. Non-progressive motilityd. Non-motile

Prevalence of male infertility:▪ 18,71 % purely asthenozoospermia▪ 63,13 % asthezoospermia associated with oligo-

and/or teratozoospermia(Curi et al., 2003)

Asthenozoospermic condition

Ethiology:

► Defect in sperm structure

► Functional deficiences

defect in mitochondrial function

► Genetic factor, etc

Anomalies in the expression of sperm protein have been found in patients

- Protamine (Mengual et al., 2003; Torregrosa et al., 2006)

- Glycoprotein 130 (GP130) (Cai et al., 2006)- 17 protein spots : ● structure-associated proteins ● metabolic enzymes (Zhao et al, 2007)

- 17 protein spots with variety function, such as for cell motiliy and proliferation, nucleosome building material (histone), etc (Martinez-Heredia et al., 2008)

DNA – Gene - Protein

Genetic factor involved in the occuring of asthenozoospermic condition

Our research:Mutation in the last four exon of

human VDAC3 gene in sperm with low motility

Mouse VDAC3 gene(+ 9,3 kb)

“VDAC3 knock-out mouse” study

Bg Nco

NeoTKBg Nco

> 70 % sperm motile in wild type

+ 17 % sperm motile in mutan (-/-)

Recombinant of mouse VDAC3 gene in

KO study

{Sampson et al., J Biol Chem, 2001}

Background of research

Aim

To analyse the last 4 exon of human VDAC3 gene in sperm with low motility

from asthenozoospermic patient

Human VDAC3 gene (10.148 bp)

6321 4 5 7 8

Examined exons

Pore-forming protein (30 - 35 kDa)

VDAC (Voltage dependent anion channel) = Porin

located in - outer mitochondrial membrane - plasma membrane

3 Isotype

(Casadio, et al., 2002) the role: - regulation ATP efflux - apoptosis

MethodsSperm from fertile men

Sperm from astheno-zoospermia patient

Sperm with normal motilitySperm with low motility

DNA isolation

PCR with specific primer for the last 4 exons of

hVDAC3 gene

Sequencing

“swim-up”

Results

PCR RESULT FOR EXON 7 OF HUMAN VDAC3 GENE

A1 A2 A3 A4 A5 A6 A7 A8 βA8 M A9 A10 A11 A12 A13 A14 A15 A16 A17 βA17

A18 A19 βA19 A20 A21 A22 A23 βA23 A24 M A25 A26 A27 A28 A29 A30 βA30 A31 A32 βA32

557 bp

557 bp

600 bp

600 bp

Note: A1 untill A32 are sperm samples from asthenozoospermia patients no 1 untill 32

Sequence analysis for Exon 6 of human VDAC3 gene

Sequence analysis of PCR product from low motile sperm

Sequence analysis of PCR product from normal motile sperm

N V D I D F S G N V D L D F S G

A L G Y K A A D F A L G Y E A A D F

Mutations in the last 4 exons of human VDAC3 gene

Sample Exon 5 Exon 6 Exon 7 Exon 8

A4 - Ile131Leu - T insertion, frameshift mutation

A5 - Ile131Leu - -

A6 - Ile131Leu - -

A7 - Ile 131Leu - -

A10 - Ile131LeuLys173Glu

- -

A13 - Ile131Leu - -

A14 - - Asp228Glu -

A17 Deletion - Deletion -

A21 - Ile131Leu - -

A23 - - Deletion -

A30 Deletion - Deletion -

A31 - - Asp228Asn -

A32 Deletion Ile131Leu Deletion Deletion

These various types of mutations in hVDAC3 gene can cause the absence of hVDAC3 channel decreased activity of the hVDAC3 channel.

It causes the retention of ATP releasing from sperm mitochondria sperm movement is destroyed.

Genetic mutations of hVDAC3 gene could be used to explain the etiology of infertile asthenozoospermic condition.

Relevance to clinical management ● Diagnostic method

1st : Semen analysis purely asthenozoospermia ?

2nd : Analysis of hVDAC3 gene at exon 5, 6, 7 and 8

DNA isolation

PCR with specific primer for the last 4 exons of hVDAC3 gene

Sequencing

“swim-up”

Sperm with low motility

Electrophoresis

No band Specific band

● Treatment for patient

Assisted reproduction, such as:

SUZI = subzonal sperm injection

ICSI = Intra cytoplasmic sperm injectionA

“Analisis molekuler pria infertil”-Mikrodelesi kromosom Y

-Analisis gen VDAC3

Departemen Biologi FKUIJl Salemba Raya No. 6 Jakarta

Telp: 021.31930379

Conclusions

• Asthenozoospemia can be caused by genetic factor

• There are mutations in the last 4 exons of human VDAC3 gene in asthenozoospermic sperm

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