Ovarian Dynamics and Follicle Differentiation; Granulosa Cell Tumors

Alan L. Johnson     Professor of Physiology Ph.D. Cornell University Postdoctoral, Cornell University

e-mail | Avianova webpage

 

Implications for the research in our laboratory pertain to both improving women’s reproductive health, together with the broader objective of understanding patterns of ovarian function and follicle development among vertebrates whose reproductive strategy consists of producing a limited number of offspring combined with considerable maternal investment (e.g., mammals, birds and a few reptiles).

Ovarian Follicle Selection

A major focus is to define the endocrine, cellular and molecular mechanisms that accomplish follicle selection, a process as yet undefined in any vertebrate. The selection of preovulatory follicles represents an event by which a species-specific number of ovarian follicles progresses from an undifferentiated to differentiated state in preparation for ovulation. This process is critical for maintaining normal reproductive cycles, and is dependent upon a balance between the maintenance of follicle viability and the loss of excessive or nonviable follicles via follicle atresia. Notably, excessive atresia can ultimately result in infertility and/or early reproductive senescence (e.g., menopause). The domestic hen represents a unique and important animal model system for such studies due to the ability to reliably identify a small cohort of prehierarchal follicles from which a single follicle per day is recruited into the preovulatory hierarchy.

The working hypothesis of our ongoing studies is that follicle selection and final differentiation prior to ovulation results from the removal of inhibitory cell signaling within the granulosa cell layer. To date, the nature of inhibition has focused on MAPK/Erk and protein kinase C (PKC) cell signaling maintained by epidermal growth factor ligands (EGFL) produced in an autocrine and paracrine fashion. The alleviation from this inhibitory signaling within the selected follicle subsequently enables FSH-induced LH receptor (LHR) induction and the initiation of steroid production. Of particular note is the recent finding that following follicle selection, MAPK/Erk and PKC signaling transitions to become obligatory for the expression and optimal activity of steroidogenic acute regulatory (STAR) protein and maximal steroidogenesis required for ovulation.

 

Simplified working model depicting the transition in cell signaling requirements from undifferentiated granulosa cells in follicles prior to selection to a differentiated state within preovulatory follicles. In undifferentiated granulosa cells one or more EGF family ligand(s) (EGFL) produced in an autocrine and/or paracrine fashion signals through ErbB1 and/or ErbB4 receptors [Woods and Johnson, 2007] to inhibit FSH-induced StAR expression and, as a result, progesterone production via activation of Erk (P-Erk) and PKC signaling. At follicle selection, granulosa cells begin the transition from FSHR dominance to LHR dominance [You et al., 1996; Johnson et al., 1996]. Cell signaling via LHR/cAMP induces StAR protein expression (and phosphorylation; not depicted) through a PKC-dependent mechanism [Woods and Johnson, 2007], enabling progesterone synthesis via P450scc activity within the mitochondria. In addition, LHR activation induces rapid transcription of one or more EGFL, which can then signal via the ErbB receptor(s) to activate the Erk signaling cascade. This LH-induced up-regulation of EGFL is mediated via PKC [Woods and Johnson, 2007]. In turn, Erk signaling is required to maintain mitochondrial electrochemical membrane potential (ψm), a prerequisite for StAR activity [Woods et al., 2007].

 

Significantly, the selective loss of ovarian follicles by atresia occurs via apoptosis and this process is first evident within the granulosa cell layer. We have established that granulosa cells from atresia-susceptible prehierarchal follicles are highly susceptible to undergoing apoptosis, in vitro. By contrast, following selection for final differentiation preceding ovulation, preovulatory follicles are not normally subject to atresia, and cultured granulosa cells from such follicles are known to be highly resistant to apoptosis. Such studies are conducted using transfection to over-express single or multiple gene products in cultured cells as well as by the targeted decrease in expression following transfection with antisense constructs. For additional information and related publications pertaining to ovarian follicle growth, development and atresia mediated via apoptosis visit our web site at: http://www.nd.edu/~avianova/. On the other hand, a decrease or absence of normal apoptotic cell death can contribute to tumor formation. Accordingly, a second and related focus utilizes human granulosa tumor cell lines to assess the role of cell survival proteins and cell death pathways in tumor formation and resistance to chemotherapeutics.

Ovarian Tumors of Granulosa Cell Origin

We are currently studying a combination of membrane receptor-mediated, cytoplasmic signaling and nuclear transcriptional events which can be linked to the development and chemotherapeutic-resistance of granulosa cell tumors. One recently investigated pro-apoptotic factor is the naturally expressed cytokine, Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL). The potential for the use of TRAIL as a chemotherapeutic, alone or in combination with conventional agents, is of considerable interest given TRAIL’s selective actions to promote apoptosis in transformed (tumor) cells, but not normal cells. These studies utilize both primary cultured granulosa cells together with cell lines established from human granulosa cell tumors.

Human granulosa tumor cell lines are currently being utilized to investigate the role of Inhibitor of Differentiation (Id) proteins as transcriptional regulators of cell proliferation, differentiation and apoptosis, within the context of cancerous cells.

 

Proposed pathway for TRAIL signaling in granulosa cells. Solid arrows depict activation of the extrinsic pathway via the death receptor, DR5/TVB. Red dotted arrows depict the predicted activation of the intrinsic pathway based upon the demonstrated expression of APAF-1 and caspase-9 mRNA in granulosa cells from both prehierarchal and preovulatory follicles. Dotted green arrows predict potential pathways by which TRAIL-induced apoptotsis is prevented, possibly via FLIPLong or decoy receptors (DcR), including osteoprotegerin (OPG).  In addition, the potential exists for TRAIL to activate the receptor adaptor protein, RIP. Because TRAIL mRNA is normally expressed within ovarian stromal, thecal and granulosa tissues, it is predicted that TRAIL can act on granulosa cells in a paracrine and/or autocrine fashion (dashed lines). aCasp., activated caspase; DD, death domain; DED, death effector domain; tBid, truncated Bid. See Johnson et al., 2007 for additional details.