טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentCohen Hadas
SubjectInvestigation of the Association between GnRH-a Sphingosine
Kinase (SphK) Activity, in Human Granulosa Cells
as a Protective Means for Decreasing the
Chemotherapy ...
DepartmentDepartment of Medicine
Supervisor Professor Zeev Blumenfeld
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

The mechanisms whereby GnRH-a decreases ovarian failure in young women exposed to gonadotoxic chemotherapy are unknown. One of the suggested possible mechanisms is the gonadal up regulation of Sphingosine-1-Phosphate[S1P], an ant apoptotic molecule.

Our aim was to examine such a possible mechanism we have evaluated the activity of Sphingosine Kinase [SK] which is the physiological enzyme generating S1P. Human granulosa cells were retrieved by follicular aspiration for IVF, after informed consent. The granulosa cell cultures [GCC] were established after separation of the GC's on Percoll and cultured in M199 with FCS and antibiotics. After 2-3 days and preincubation in serum free medium, the GC's were incubated with native GnRH, GnRH-agonist, GnRH-antagonist, dimethyl-sphingosine, or control medium. After 24 hours, the cells were trypsinized, lysed, and the SK activity was determined by conversion of added Sphingosine to SK followed by TLC separation with radioactive P32, by phosphoimaging. ATP- P32 was added for labeling SK, by the method of Sara Spiegel [Richmond, VA, USA].

There was no significant change in SK activity following incubation with either GnRH or its analogues. Neither the agonist, nor the antagonist or native GnRH affected SK activity. However, dimethyl-sphingosine, an inhibitor of SK, decreased its activity.

GnRH-a does not increase the activity of SK in GCC, in vitro. However, S1P may still be possibly involved in the protective mechanism of GnRH-a against chemotherapy associated gonadotoxicity through an inhibitory effect on S1P-phosphatase.