Ph.D Thesis

Ph.D StudentRegev Avital
SubjectThe Semaphorin-plexin Ligand-Signaling System in Rodent and
Human Ovary
DepartmentDepartment of Medicine
Supervisor PROFESSOR EMERITUS Eliezer Shalev
Full Thesis textFull thesis text - English Version


Plexin-Semaphorin system originally found in the nervous system, are also implicated in cardiac, skeletal and immune response and involved in morphogenesis, angiogenesis, tumor growth and metastasis via cytoskeleton reorganization.

Expression, role and mechanism of action of Plexin-B1 and Semaphorin-4D (Sema-4D) in the mouse ovary and the expression of Plexin-B1 in human granulosa cells obtained from normal ovulatory and PCO patients as example for pathologic follicular growth were studied.

Expression and localization were tested by immunohistochemistry, Western Blot and RT-PCR. Role on follicular development was examined by in vitro growth of primordial follicles in the presence or absence of neutralizing antibodies against Plexin-B1 or Sema-4D. Follicular growth, morphology and steroid hormone secretion were tested. The effect of neutralizing antibodies against Plexin-B1 or Sema-4D  on f-actin formation and GTPases R-RAS and Rho-A were studied using immunofluorescent. Plexin-B1 expression in human granulosa cells obtained from normal ovulatory and PCO patients was tested by immunohistochemistry and RT-PCR. Immunofluorescent staining for F-actin was performed as well.

Plexin-B1 and Sema-4D are expressed in the mouse ovary mostly in the granulosa cells and their expression is hormonal regulated. In vitro model of follicular growth in the presence of neutralizing antibodies against plexin-B1 caused dense follicles with reduced diameter and low steroids hormones secretion. In the presence of Sema-4D normal follicular morphology was observed with constant increase in diameter and elevated steroids hormone secretion compared with control. This effect was abolished after addition of the antibodies. Sema-4D induced cell collapse manifested as reduction in f-actin formation. This modulation involves changes in R-RAS and Rho-A distribution. The effect of Sema-4D on f-actin and small GTPases in the growing follicle is dependent on follicular development stage. No change between normal-ovulatory and PCO granulosa cells was found at the transcript level of Plexin-B1, however, f-actin formation was different between the two groups.

Plexin-B1 plays a role in follicular growth and morphology, most likely through regulation of f-actin fiber organization via small GTPases Rho-A and R-Ras signal transduction.

Cyclic changes in R-RAS and Rho-A distribution lead to cytoskeltal changes of genes and proteins as reflected by the reduction in f-actin formation. Normal Semaphorin-4D-Plexin-B1 signaling will be followed by cell collapse leading to granulosa cells proliferatation, migration ending with grown and re-shape preovulatory follicle. Changes in granulosa cell morphology modulate steroid secretion. Disturbed Plexin-B1 signaling will results with unbalanced GTPases distribution which hampers cytoskeltal protein production resulting in abnormal follicle development.