Regulation of apoptosis in the female reproductive system

Tommi Vaskivuo

Department of Obstetrics and Gynaecology, University of Oulu

Abstract

Apoptosis is a genetically programmed mechanism for a multicellular organism to remove cells that are unnecessary, or potentially harmful. The female reproductive system is characterised by a high rate of cellular proliferation. At the same time, apoptosis is also abundant during the normal physiological function of the ovary and endometrium. More than half of the 7 million oocytes that are produced during human ovarian development are deleted before birth and only about 400 oocytes reach the stage of ovulation during the female fertile lifespan. The fate of the non-ovulatory follicles is atresia, occurring through the mechanism of apoptosis. The endometrium goes through radical renewal processes during each menstrual cycle. Apoptosis has been suggested to participate in the regulation of endometrial cellular homeostasis. Errors in this mechanism can result in endometrial diseases such as hyperplasia and cancer. In this work, apoptosis and its regulation were studied in the human fetal and adult ovary, normal endometrium and endometrial pathologies.

In fetal ovaries, apoptosis was already abundantly present in oocytes at 13 weeks of gestation. The maximum rate of apoptosis was seen between the 14th and 20th weeks, after which apoptosis decreased towards term. Ovarian Bcl-2 expression was detected in early fetal life during weeks 13 and 14. Bax expression was observed throughout the studied period, from week 13 to 40. The expression of transcription factor GATA-4, which is linked to follicular survival, was localised to the granulosa cells and was high in early fetal life and decreased somewhat towards term. In adult life apoptosis was located in the granulosa cells of the growing follicles. In ovarian biopsies from women homozygous for the inactivating C566T mutation of the FSH receptor, apoptosis or GATA-4 expression was not detected. During corpus luteum regression a peak in apoptosis was detected 10–12 days after the LH surge, and was preceded by an increase in 17HSD type 1 and TNF-_ expression. During normal menstrual cycles, the highest rate of apoptosis was observed in the menstrual endometrium. This increase in apoptosis was preceded by a decreased Bcl-2/Bax ratio. In endometrial hyperplasia, the rate of apoptosis was similar to that seen during normal proliferation of the endometrium, but an apparent increase was observed in grade II endometrial carcinoma. In grade III carcinoma, the rate of apoptosis was lower than in grade II carcinoma but higher than in hyperplasia.

These results indicate that apoptosis is the mechanism behind the substantial oocyte demise during ovarian development. During adult life, apoptosis was mainly localised to the granulosa cells of the growing follicles which do not reach the stage of a dominant follicle. In ovaries where FSH action is abolished, folliculogenesis was impaired and ovarian apoptosis was negligible. Apoptosis is also the underlying mechanism of corpus luteum regression. In the endometrium, apoptosis has a role in rejuvenating the endometrium for growth during the next endometrial cycle and in regulating cellular homeostasis.

Table of Contents
Acknowledgements
Abbreviations
List of original publications
1. Introduction
2. Review of the literature
2.1. Apoptosis
2.1.1. History
2.1.2. Apoptosis vs. necrosis
2.2. Common mechanisms of apoptosis
2.2.1. Regulation of apoptosis
2.2.2. Execution of apoptosis
2.2.3. Role of apoptosis in development
2.2.4. Apoptosis and tissue homeostasis
2.2.5. Apoptosis in disease
2.2.6. Studying apoptosis
2.3. Ovarian function
2.3.1. Ovarian development
2.3.2. The adult ovary
2.3.3. Regulation of ovarian apoptosis
2.4. Endometrial function
2.4.1. Uterine development
2.4.2. Regulation of endometrial function during the menstrual cycle
2.4.3. Endometrial cancer and hyperplasia
3. Aims of the study
4. Materials and methods
4.1. Tissue samples
4.1.1. Fetal and neonatal ovaries
4.1.2. Adult ovaries
4.1.3. Patients with inactivating FSHR mutation
4.1.4. The corpus luteum
4.1.5. Cycling endometrium
4.1.6. Endometrial hyperplasias and carcinomas
4.2. Cell culture
4.3. In situ 3’ end labelling of apoptotic cells
4.4. Gel electrophoretic DNA fragmentation analysis
4.4.1. Radioactive DNA fragmentation analysis
4.4.2. Non-radioactive DNA fragmentation analysis
4.5. In situ hybridisation analysis
4.5.1. Bcl-2 and Bax
4.5.2. GATA-4
4.5.3. 17HSD type 1 and 2
4.6. Northern blotting
4.7. Immunohistochemistry
4.8. Western blotting
4.9. FSH and hCG stimulation
4.10. Hormone measurements
4.11. Histopathological analysis
4.12. Statistics
5. Results
5.1. Apoptosis in the fetal ovary
5.1.1. Regulation of apoptosis in fetal ovary
5.2. Apoptosis in adult ovary
5.2.1. Apoptosis-regulating factors in adult ovary
5.3. Role of FSH in ovarian apoptosis
5.4. Apoptosis in the corpus luteum
5.4.1. Regulation of apoptosis in the corpus luteum
5.4.2. 17HSD type 1 and 2 expression in the corpus luteum
5.5. Apoptosis in the endometrium during the menstrual cycle
5.5.1. Regulation of apoptosis in cycling endometrium
5.5.2. Proliferation during normal endometrial cycles
5.6. Apoptosis in endometrial hyperplasias and carcinomas
5.6.1. Apoptosis in endometrial hyperplasia
5.6.2. Apoptosis in endometrial adenocarcinoma
5.7. Functional study of C566T FSHR mutation
6. Discussion
6.1. Germ cell attrition
6.2. Follicular atresia
6.3. Two-cell-two-gonadotrophin theory
6.4. Corpus luteum regression
6.5. Endometrial apoptosis during the normal menstrual cycle
6.6. Regulation of endometrial homeostasis
6.7. Future perspectives
7. Summary and conclusions
References
List of Tables
1. Mouse models of ovarian failure.
2. Transgenic and knockout animal models for abnormal ovarian cell death.
3. Materials and methods of the studies.
4. Antibodies.
5. Main results of the studies.
6. Effects of treatment with FSH and/or hCG on serum inhibin B (ng/l), T (nmol/l) and E2 (nmol/l) concentrations in subjects A, B and C (sex/treatment) homozygous for C566T FSHR mutation
List of Figures
1. Two cell death pathways, necrosis and apoptosis. Necrosis involves breakdown of the cellular membrane, which leads to leakage of intracellular proteins to the extracellular space and subsequently, inflammation. Necrosis usually affects large groups of cells while apoptosis typically involves single cells that undergo organised destruction of the cellular cytoskeleton and formation of apoptotic bodies, which are phagocytosed without an inflammatory reaction.
2. Conserved cell death programme from worms to humans. Analogues of Ced genes that control apoptosis in C. elegans have been found in humans. Death signals initiate the pathway by directly inhibiting the actions of anti-apoptotic proteins (Ced-9 in C. elegans and Bcl-2 in humans) or by activating factors that are capable of suppressing the actions of these proteins (such as Egl-1 and BAD). Inhibition of Ced-9 or Bcl-2 leads to triggering of the next step in the suicide programme. Subsequent activation of Ced-4 or Apaf-1 factors sets off the final executors of apoptosis, Ced-3 or Caspases.
3. Four stages leading to apoptosis according to Morita and Tilly (Morita & Tilly 1999.) The first stage comprises of different potentially harmful stimuli that interact with a cell. In the second stage, an early signalling molecule is activated. This signal is processed by a regulatory mechanism, which evaluates the strength of the apoptosis inducing signal against anti-apoptotic signals in the third stage. If the death inducers prevail the cell commits to apoptosis and enters the fourth and final stage where specific executor proteins are responsible for the organized destruction of the cell.
4. Regulation of death receptor signalling. Activated Fas trimer recruits an adaptor protein called FADD, through interaction with their respective death domains (DDs). FADD functions as a bridge between Fas and downstream signal transduction, which is mediated by the N-terminal region of the protein, termed death effector domain (DED). The TNFR1 mediated pathway utilizes TRADD protein in recruitment of FADD. The binding of pro-caspase-8 to the FAS/FADD or alternatively the TNFR1/TRADD/FADD complex activates autoprocessing of the pro-enzyme to its active form. A mutated form of caspase-8, c-FLIP, can also bind to FADD, thus inhibiting the binding and activation of caspase-8. Activated caspase-8 is able to induce apoptosis through a mitochondrial pathway by cleaving BID, or directly by activating downstream effector caspases. Mitogens and growth factors can directly inhibit the activation of caspase-8 through an unknown pathway. Additionally, activation of MAPK/ERK pathway can lead to phosphorylation, i.e. inactivation of BAD. TNFR1 also utilizes an anti-apoptotic signalling pathway. TRAF2 can bind to TNFR1/TRADD complexes and activate a pathway that leads to phosphorylation of Iκ B and consequently activation of transcription factor NF-κ B. The TNFR1/TRADD/TRAF2 complex can also recruit a fourth protein, termed RIP, which possesses a serine-threonine kinase domain with unknown function.
5. The Bcl-2 family. The bcl-2 gene encodes a 25–26 kDa protein that bears no obvious structural clues to suggest the mechanism by which it controls apoptosis. It has a hydrophobic transmembrane domain (TM) of 21 amino acids in its C-terminus that enables the insertion of the protein into membranes. Bcl-2 also contains 4 conserved homology regions, termed Bcl-2 homology domains 1, 2, 3 and 4 (BH1, BH2, BH3 and BH4). Most of the Bcl-2 family members possess the TM region and variable amounts of BH regions. Through interactions of these BH domains, members of the Bcl-2 family can form homo- and heterodimers with each other and apparently titrate one another’s functions.
6. Representation of possible interactions between anti-apoptotic (white) and pro-apoptotic (black) members of the Bcl-2 family at the outer mitochondrial membrane. Apoptotic signals relocate Bax from the cytoplasm to the mitochondrion (1). Anti-apoptotic members of the Bcl-2 family, such as Bcl-2 itself and Bcl-XL can block the pro-apoptotic effects of Bax by binding it and forming heterodimers (2). However, other pro-apoptotic Bcl-2 proteins, e.g. BAD and BID, can interact with Bcl-2 and Bcl- XL and prevent their anti-apoptotic function (3). Eventually, the relationship between pro-apoptotic and anti-apoptotic factors determines the susceptibility to apoptosis. If there are more pro-apoptotic factors, the mitochondrion subsequently loses its membrane potential and a number of apoptosis- promoting molecules, such as cytochrome c and apoptosis-inducing factor (AIF) are released into the cytoplasm (4).
7. Activation of apoptosis through mitochondrial pathway. Extracellular signals can have an effect on the relationship of Bcl-2 family members at the surface of mitochondria. Pro-apoptotic Bcl-2 proteins can release a variety of molecules from the mitochondrial compartment. Cytochrome c is considered to be the primary mitochondrial factor in caspase-mediated apoptosis. Together with Apaf-1 and procaspase-9, cytochrome c forms the apoptosome, which is a potent activator of caspase-3. Smac/Diablo is a mitochondrial factor that can inhibit the action of IAP proteins, which themselves can prevent caspase-3 activation and action. AIF is also released from mitochondria and it can activate apoptosis via unknown, caspase-independent pathway.
8. Germ cell attrition and follicular atresia according to Kaipia & Hsueh 1997. Germ cells migrate to the ovary during early embryonic development. Their number increases through mitotic divisions but most of the oocytes formed during development do not survive to the time of birth.
9. Folliculogenesis and classification of growing follicles in the human ovary according to Gougeon (1996). Growing follicles enter class 2 usually in the late luteal phase, class 3 between late luteal and early follicular phases, class 4 during late follicular phase and become recruitable class 5 follicles during late luteal phase.
10. Two-cell-two-gonadotrophin theory. LH induces androstenedione synthesis in theca cells. Driven by the FSH stimulus, granulosa cells process androstenedione into estrone which is further converted into estradiol by type 1 17HSD enzyme.
11. Changes in the endometrial tissue and hormone values during the menstrual cycle.
12. Presentation of apoptosis in fetal ovaries. Apoptosis was mainly detected in oocytes and only rarely were apoptotic stromal or granulosa cells seen. The rate of apoptosis remained high from the 13th week to the 20th week and decreased thereafter. At term no apoptotic oocytes were detected.
13. Apoptosis in adult ovaries. Apoptosis was mainly observed in the granulosa cells of growing follicles, but not in oocytes. The figure displays the percentage of follicles that contained apoptotic granulosa cells.
14. DNA fragmentation analysis of apoptotic granulosa cells from three different IVF patients. Each lane represents granulosa cells gathered from a single follicle.
15. Graphical presentation of apoptosis in the corpus luteum during luteal regression. CL age is presented as days after the LH surge.
16. Presentation of apoptosis in the endometrium during the menstrual cycle in endometrial glands. EP = early proliferative (cycle days 3–9), LP = late proliferative endometrium (10–14), ES = early secretory (15­–19), MS = middle secretory (20–26), LS = late secretory endometrium (27–29), ME = menstruating endometrium (1–5)
17. Relationship of Bcl-2 and Bax proteins in the endometrium during the menstrual cycle. EP = early proliferative, LP = late proliferative endometrium, ES = early secretory, MS = mid- secretory, LS = late secretory endometrium.
18. Apoptotic cells in endometrial glands in normal endometrium, endometrial hyperplasia and cancer.
19. Representation of ovarian apoptosis in fetal and adult life. In fetal ovaries apoptosis is mainly observed in the oocytes and the majority of them are deleted before birth. It is likely that a low rate of oocyte apoptosis also continues in postnatal life. However, in adult ovaries, apoptosis in only detected in the granulosa cells of growing follicles.
20. Model of corpus luteum regression. High 17HSD1 expression was observed in the mid-luteal phase. The resulting high local E2 concentration can have a role in predisposing the CL to apoptosis. In the late luteal phase increase in TNF-_ expression can function as a trigger to apoptosis.
21. Regulation of apoptosis in human endometrium. In secretory endometrium Bcl-2/Bax ratio decreases. This event is likely to be under control of ovarian hormones. The decrease in Bcl-2/Bax ratio precedes the increase of apoptosis in the menstruating endometrium and it is possible that other factors play a role in induction of apoptosis during menstruation.
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