Asmarinah

1. UNIVERSITY OF INDONESIA FACULTY OF MEDICINE DEPARTMENT OF MEDICAL BIOLOGY Genetic factor in asthenozoospermic patient: relevance to laboratorium test and clinical management Asmarinah

2. 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 motility c. Non-progressive motility d. Non-motile

3. Asthenozoospermic condition Prevalence of male infertility: ▪ 18,71 % purely asthenozoospermia ▪ 63,13 % asthezoospermia associated with oligo- and/or teratozoospermia (Curi et al., 2003) Ethiology: ► Defect in sperm structure ► Functional deficiences  defect in mitochondrial function ► Genetic factor, etc

4. 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)

5. DNA – Gene - Protein

6. Our research: Mutation in the last four exon of human VDAC3 gene in sperm with low motility Genetic factor involved in the occuring of asthenozoospermic condition

7. Bg Nco Bg Nco TK Neo Background of research “VDAC3 knock-out mouse” study Mouse VDAC3 gene (+ 9,3 kb) Recombinant of mouse VDAC3 gene in KO study > 70 % sperm motile in wild type + 17 % sperm motile in mutan (-/-) {Sampson et al., J Biol Chem, 2001}

8. Human VDAC3 gene (10.148 bp) 1 2 3 4 5 6 7 8 Aim To analyse the last 4 exon of human VDAC3 gene in sperm with low motility from asthenozoospermic patient Examined exons

9. VDAC (Voltage dependent anion channel) = Porin • Pore-forming protein (30 - 35 kDa) • located in - outer mitochondrial membrane - plasma membrane • 3 Isotype • the role: - regulation ATP efflux - apoptosis (Casadio, et al., 2002)

10. Sperm with low motility “swim-up” DNA isolation PCR with specific primer for the last 4 exons of hVDAC3 gene Sequencing Methods Sperm from astheno- zoospermia patient Sperm from fertile men Sperm with normal motility

11. Results

12. A1 A2 A3 A4 A5 A6 A7 A8 βA8 M A9 A10 A11 A12 A13 A14 A15 A16 A17 βA17 557 bp 557 bp 600 bp 600 bp A18 A19 βA19 A20 A21 A22 A23 βA23 A24 M A25 A26 A27 A28 A29 A30 βA30 A31 A32 βA32 PCR RESULT FOR EXON7 OF HUMAN VDAC3 GENE Note: A1 untill A32 are sperm samples from asthenozoospermia patients no 1 untill 32

13. Sequence analysis for Exon 6 of human VDAC3 gene Sequence analysis of PCR product from normal motile sperm Sequence analysis of PCR product from low 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

14. Mutations in the last 4 exons of human VDAC3 gene

15. 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.

16. Relevance to clinical management ● Diagnostic method 1st : Semen analysis  purely asthenozoospermia ? 2nd : Analysis of hVDAC3 gene at exon 5, 6, 7 and 8 Sperm with low motility “swim-up” DNA isolation PCR with specific primer for the last 4 exons of hVDAC3 gene Electrophoresis No band Specific band Sequencing

17. ● 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 FKUI • Jl Salemba Raya No. 6 Jakarta • Telp: 021.31930379

18. Conclusions • Asthenozoospemia can be caused by genetic factor • There are mutations in the last 4 exons of human VDAC3 gene in asthenozoospermic sperm

19. References 1. Asmarinah, Hinsch KD, Aires VA, Hinsch E. Effect of anti-porin type2 antibodies upon bovine sperm motility and acrosomal status. Andrologia 2003 (35):2. 2. Bourgeron T. Mitochondrial function and male infertility. Results Probl Cell Differ 2000 (28):187-210. 3. Hinsch KD, de Pinto V, Aires VA, Schneider X, Messina A, Hinsch E. Voltage-dependent anion-selective channels VDAC2 and VDAC3 are abundant protein in bovine outer dense fiber, a cytoskeletal component of the sperm flagellum. J Biol Chem 2004 279(15):15281-8. 4. Huynh T, Mollard R, Trounson A. Selected genetic factors associated with male infertility. Hum Reprod update 2002 (8):183-198. 5. Lemasters JJ, Holmuhamedov E. Voltage-dependent anion channel (VDAC) as mitochondrial governator--thinking outside the box. Biochim Biophys Acta. 2006 1762(2):181-90. 6. Rostovtseva T, Colombini M. VDAC channels mediate and gate the flow of ATP : Implications for the regulation of mitochondrial function. Biophys J 1997 (72):1954- 1962. 7. Sampson MJ, Decker WK, Beaudet AL, et al. Immotile sperm and infertility in mice lacking mitochondrial voltage-dependent anion channel type 3. J Biol Chem 2001 (276):39206-11.