Alternative pathways of ovarian apoptosis: death for life

Abstract

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few, but healthy embryos. Degeneration of the old corpora lutea in each estrus/menstrual cycle by programmed cell death is essential for maintaining the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, play an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows for systematic studies of the mechanisms that control steroidogenesis and apoptosis of granulosa cells. We have discovered that during initial stages of granulosa cell apoptosis progesterone production does not decrease. In contrast, we found that it is elevated for up to 24 hr following the onset of the apoptotic stimuli exerted by starvation, cAMP, p53 or tumor necrosis factor α stimulation, before total cell collapse. These observations raise the possibility for an alternative unique apoptotic pathway, one that does not involve mitochondrial cytochrome C release associated with the destruction of mitochondrial structure and steroidogenic function. Using mRNA from apoptotic cells and Affymetrix DNA microarray we discovered that Granzyme B, a protease that normally resides in T cytotoxic lymphocytes and natural killer cells of the immune system is expressed and activated in granulosa cells, thereby allowing the apoptotic signals to bypass mitochondrial signals for apoptosis, which can preserve their steroidogenic activity until complete cell destruction. This unique apoptotic pathway assures the cyclicity of estradiol and progesterone release in the estrus/menstrus cycle even during the initial stage of apoptosis.

Introduction

Ovarian cell death is a crucial event in maintaining ovarian homeostasis in mammals. It ensures that in every estrus/menstrus cycle only one or very few follicle-enclosed oocytes will reach the stage of a Graafian follicle and will ovulate. This is essential in preventing multiple embryos during pregnancy. The rest of the follicles are gradually eliminated during the fertility period of the female. The apoptotic process of the old corpora lutea is essential for preserving the cyclicity and for ensuring the release of progesterone during the estrus/menstrus cycle (reviewed in [1], [2], [3], [4], [5]). There are several factors that may control apoptosis of granulosa cells. In each stage of the cycle about 50% of the large preantral and antral follicles are in the process of apoptotic death [6]. In most antral follicles apoptosis is initiated at inner layers of the membrana granulosa, bordering the follicle antrum (Fig. 1). Apoptosis is protected by the basement membrane which can sequester bFGF where both laminin, the main component of the basement membrane, and bFGF serve as survival factors [7], [8]. Interestingly, laminin protects against apoptosis without affecting steroidogenesis, while bFGF enhances formation of progesterone in preovulatory follicular cells [7].

Induction of apoptosis in vivo by hypophysectomy of female rats surprisingly elevates progesterone production dramatically in the ovary [9]. This may suggest that the initial steps of apoptosis enhance rather than block progesterone production. A similar phenomenon is found upon induction of apoptosis in highly luteinized granulosa cells [10], [11], [12] (Fig. 2) which raises the question: can steroidogenesis and apoptosis exist in the same cell? If the answer is positive, then the next question is how mitochondrial integrity, which is the prime target of apoptotic signals in numerous cell types, is preserved during the initial steps of apoptosis in ovarian steroidogenic cells?