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

InfertilityInfertility

* 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.

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

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.

Small colony Medium colony Large colony

ACB

Murine colony formation in agar4 weeks culture

Development of murine sperm-like cells in agar

Small Medium Large

Colony formation in agar/methylcellulose

Non human primate (NHP) system

GFR- SALL-4 VASA

CREM-1NC

Expression of spermatogenesis markers in juvenile NHP testicular germ cells cultured in methylcellulose

ACROSIN

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

Immunofluorescence staining of spermatogenesis markers from prepubertal boys before and after 5 weeks of culture

Pre-meiotic (SSC)Protamine

Post-meioticMeioticLDH

CD-9GFR-C-Kit

Pre-meiotic markers (SSC)Before

After

CD-9 GFR-

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

Pre-meiotic

(SSC)

Meiotic Post-meiotic

Weeks C-kit GFR- CD9 CREM LDH Protamine Acrosin

Before

culture0 - + - - - - -

After

culture5 - + _ - + - +

RNA expression levels of spermatogenic markers in colonies developed in methylcellulose from cells isolated from

biopsies of adult azoospermic patients