1. MALE INFERTILITY Development of in vitro spermatogenesis and genetic analysis Huleihel Mahmoud, Ph.D The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev

2. Infertility * Infertility affects 1/5 couples of reproductive age and in half male infertility is the sole or a contributory cause. *Testicular disorders affecting spermatogenesis account for 2/3 of cases and the majority are of unknown etiology. * Genetic and epigenetic factors are involved in human infertility. Appromimately10% are defined.

3. Spermatogenic Dysfunction Possible Etiologies: 1.Congenital (inherited, de novo, etc.) 2.Acquired (infection, environmental factors, etc.) 3.Iatrogenic (chemotherapy, radiotherapy, etc.) There has been a gradual decrease in male fertility world wide

4. Goals We propose to address testicular dysfunction by: 1. Identifying specific genes that can contribute to spermatogenic dysfunction. 2. Developing new in vivo and in vitro technologies that enable propagation and differentiation of spermatogonial stem cells (SSCs) into functional sperm.

5. Small colonyMedium colonyLarge colony A C B Murine colony formation in agar 4 weeks culture

6. Development of murine sperm-like cells in agar

7. Small MediumLarge Colony formation in agar/methylcellulose Non human primate (NHP) system

8. GFR-  SALL-4 VASA CREM-1 NC Expression of spermatogenesis markers in juvenile NHP testicular germ cells cultured in methylcellulose ACROSIN

9. Growth of prepubertal (7 y) testicular cells (germ cells) in culture (2 weeks)

10. Immunofluorescence staining of spermatogenesis markers from prepubertal boys before and after 5 weeks of culture Pre-meiotic (SSC) Protamine Post-meioticMeiotic LDH CD-9 GFR-  C-Kit Pre-meiotic markers (SSC) Before After CD-9 GFR- 

11. Development of colonies from cells isolated from biopsies of adult azoospermic patients

12. Post-meioticMeioticPre-meiotic (SSC) AcrosinProtamineLDHCREMCD9 GFR-  C-kitWeeks -----+-0 Before culture +-+-_+-5 After culture RNA expression levels of spermatogenic markers in colonies developed in methylcellulose from cells isolated from biopsies of adult azoospermic patients