Mol. Hum. Reprod. Advance Access originally published online on April 15, 2005
Molecular Human Reproduction 2005 11(5):319-324; doi:10.1093/molehr/gah168
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Insulin and insulin-like growth factors inhibit and luteinizing hormone augments ovarian theca-interstitial cell apoptosis
1Department of Gynecology and Obstetrics, Division of Infertility and Reproductive Endocrinology, Poznan University of Medical Sciences, 60-535 Poznan, Poland, 2Department of Obstetrics and Gynecology, Vincent Center for Reproductive Biology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114, USA and 3Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06510, USA
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. Email: antoni.duleba{at}yale.edu
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Abstract |
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Theca-interstitial (T-I) cells play a fundamental role in the control of ovarian function. Steroidogenic activity and growth of the T-I cells are regulated by many paracrine and endocrine factors. However, little is known about the mechanisms controlling T-I death. In an in vitro model of apoptosis, purified rat T-I cells were cultured for 24 h with serum and subsequently for up to an additional 24 h with serum or in serum-free medium with or without insulin, insulin-like growth factors (IGF-I and IGF-II) and LH or 8-bromo-cyclic AMP (8Br-cAMP). Apoptosis was identified by histological assessment of nuclear morphology and by detection of internucleosomal cleavage and quantified by determination of [
32P]-dideoxy-ATP 3'-end labeling of low molecular weight DNA. Serum withdrawal resulted in nuclear condensation and fragmentation into apoptotic bodies of T-I cells and led to pronounced DNA cleavage. Insulin (10 nM) protected T-I cells from apoptosis, reducing DNA fragmentation by 39 ± 8% compared to serum-free controls. IGF-I (10 nM) and IGF-II (10 nM) had comparable antiapoptotic effects, decreasing DNA fragmentation by 55 ± 9% and 37 ± 14%, respectively. In contrast, LH (100 ng/ml) and 8Br-cAMP (1 mM) augmented the pro-apoptotic effect of serum withdrawal, increasing DNA fragmentation by 85 ± 55% and 72 ± 42%, respectively. The antiapoptotic effects of insulin and IGFs and the pro-apoptotic effect of LH, acting via the cAMP system, may be important in the maintenance of T-I homeostasis. Moreover, excessive levels of insulin and free IGFs may lead to T-I cell hyperplasia characteristic of conditions such as polycystic ovary syndrome. Key words: apoptosis/insulin/insulin-like growth factors/LH/ovary/theca-interstitial cells
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Introduction |
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Theca-interstitial (T-I) cells play an essential role in the maintenance of ovarian integrity and function. Precise regulation of the size of the T-I compartment is imperative for ovarian homeostasis and depends on the balance between cell proliferation and death. Dysregulation of the T-I growth and differentiation, resulting in hyperplasia of T-I cells, may contribute to the development of pathological conditions such as polycystic ovary syndrome (PCOS) or hyperthecosis. The role of LH, insulin and insulin-like growth factors (IGF-I and II) as agents controlling T-I steroidogenesis is well established (Cara and Rosenfield, 1988
; Caubo et al., 1989). We have also shown that these factors participate in the regulation of proliferation of T-I cells (Duleba et al., 1997, 1999).
Earlier investigations have indicated that follicles undergo atresia by the process of programmed cell death (Hughes and Gorospe, 1991; Tilly et al., 1991; Kaipia and Hsueh, 1997). Apoptosis was identified not only in granulosa but also in theca cells (Tilly et al., 1992; Palumbo and Yeh, 1994; Zerbinatti et al., 2001). During follicular atresia almost an entire population of granulosa cells dies by apoptosis, whereas not all T-I cells undergo apoptotic death. A subpopulation of T-I cells undergoes hypertrophy and is incorporated into the ovarian interstitium (Erickson et al., 1985; Palumbo and Yeh, 1994). However, in contrast to the present extensive literature on the regulation of granulosa cell death (Tilly and Robles, 1999), far less is known about mechanisms controlling the death of T-I cells. A family of transforming growth factors was recently demonstrated to participate in the regulation of T-I cells apoptosis in vitro (Foghi et al., 1997; Pehlivan et al., 2001). Moreover, a Fas–Fas ligand system was localized in T-I cells and was demonstrated to activate execution of programmed cell death in cultured T-I cells (Kondo et al., 1996; Foghi et al., 1998; Porter et al., 2000).
Studies on the endocrine regulation of T-I programmed cell death in vivo are problematic owing to the presence of a heterogeneous population of ovarian follicles and the inability to evaluate individual intra-ovarian survival and death factors. We have recently demonstrated that serum deprivation induces internucleosomal DNA cleavage in cultured T-I cells and we developed an in vitro model to study apoptotic death in purified rat T-I cells (Pehlivan et al., 2001). The aim of the present study was to demonstrate that the serum withdrawal induces characteristic morphological features of apoptosis in T-I cells and to evaluate the effects of insulin, IGF-I, IGF-II and LH/8Br-cAMP on rat T-I cell death.
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Materials
The following materials were purchased from Sigma Chemical Co. (St Louis, MO, USA): Medium 199 with Hank's Balanced Salt Solution (HBSS) and sodium bicarbonate, Medium 199 with HBSS (x10), McCoy's 5a medium (modified, with sodium bicarbonate), L-glutamine, bovine serum albumin (BSA), fetal bovine serum (FBS), 17ß-estradiol (E2), sesame oil, Percoll, paraformaldehyde, 4',6-diamidino-2-phenylindole (DAPI), transfer RNA, 8-bromo-cyclic AMP (8Br-cAMP), bovine insulin, human recombinant IGF-I and IGF-II. Collagenase type I (Clostridium histolyticum, CLS1; 146 U/mg) and deoxyribonuclease I (bovine pancreas; 2298 U/mg) were obtained from Worthington Biochemical Co. (Freehold, NY, USA). The following materials were purchased from Grand Island Biological Co. (Grand Island, NY, USA): trypan blue stain (0.4%; wt/vol), antibiotic–antimycotic preparation (penicillin, 10 000 IU/ml; streptomycin, 10 000 mg/ml; amphotericin B, 25 mg/ml) and Dulbecco's phosphate buffered saline (PBS, x1, pH 7.2, without MgCl2 and CaCl2). HEPES was purchased from American Bioanalytical (Natick, MA, USA). The National Hormone and Pituitary Program, NIDDK and Dr A.F. Parlow (Bethesda, MD, USA) donated the Ovine LH (o-LH-26). Terminal deoxynucleotidyl transferase (25 U/µl), 5x concentrated Tdt enzyme reaction buffer, cobalt chloride (25 mM) and DNAase free RNAase (500 µg/ml) were obtained from Boehringer Mannheim Corp. (Indianapolis, IN, USA). [
32P] dideoxy-ATP was purchased from Amersham Life Science Inc. (Arlington Heights, IL, USA).
Isolation of T-I cells
Ovarian T-I cells were obtained from immature (25 days old) female Sprague–Dawley rats (Taconic Farms, Germantown, NY, USA) injected with 17ß-E2 (1 mg/0.3 ml sesame oil s.c.) from 28 to 30 days of age. On day 31 of age the animals were anesthetized with ketamine and xylazine (i.p.) and sacrificed by perfusion with 0.9% saline. All treatments and procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and a protocol approved by the Yale University Animal Care Committee. Ovaries were dissected and T-I cells were purified using discontinuous Percoll gradient centrifugation as described previously (Magoffin and Erickson, 1988; Duleba et al., 1997). The cells were counted and viability determined with the trypan blue stain exclusion test was done routinely in the 85–95% range. The purity of fresh T-I cell preparations has been evaluated histochemically as described previously. Preparations of T-I cell stained positive for vimentin in 95.4 ± 0.7% (mesenchymal marker), for cytokeratin in 7.6 ± 1.6% (epithelial marker) and for factor VIII in 2.2 ± 0.7% (endothelial marker) (Duleba et al., 1997).
T-I cell culture
For morphological analysis of nuclear and cellular morphology, cultures of T-I cells were grown in Lab-Tek 2 chamber polystyrene tissue culture slides (Miles Scientific Inc., Naperville, IL, USA) at a density of approximately 2 x 106 cells/well for 24 h at 37°C in an atmosphere of 5% CO2 in humidified air in McCoy's 5a medium with penicillin (10 000 IU/ml), streptomycin (10 000 mg/ml), amphotericin B (25 mg/ml), 0.1% BSA and 2 mM L-glutamine supplemented with 10% FBS. Subsequently, the cells were washed and re-incubated in serum-free McCoy's 5a medium with or without 10% FBS for additional 24 h.
For subsequent DNA extraction T-I cells were incubated in 6-well plates (Falcon, Becton Dickinson Labware, Lincoln Park, NJ, USA) at a density of approximately 106 cells/well for 24 h at 37°C in an atmosphere of 5% CO2 in humidified air in McCoy's 5a medium with penicillin (10 000 IU/ml), streptomycin (10 000 mg/ml), amphotericin B (25 mg/ml), 0.1% BSA, 2 mM L-glutamine supplemented with 10% FBS. Subsequently, the cells were washed and only viable T-I cells, attached tightly to the culture plates, were left. These T-I cells were re-incubated in serum-free McCoy's 5a medium with or without FBS (10%), insulin (10 nM), IGF-I (10 nM), IGF-II (10 nM), LH (100 ng/ml) and 8Br-cAMP (1 mM) for an additional 6–24 h.
Histological assessment of apoptotic death of T-I cells
At the end of the incubation period, cells were immediately fixed with 2% neutral-buffered para-formaldehyde for 30 min at 4°C and stained with DNA-binding fluorescent dye—DAPI, at a final concentration of 20 µg/ml for 15 min at room temperature in the dark. Fluorescence microscopy with an ultraviolet light filter was performed to evaluate samples. Morphological criteria of apoptosis described previously were applied (Huppertz et al., 1999; Stadelmann and Lassmann, 2000). Findings of nuclear condensation (brightly fluorescent pyknotic nuclei) and multiple, densely stained chromatin fragments of nearly spherical shape (apoptotic bodies) were used for defining apoptotic cells.
DNA extraction
At the termination of culture, genomic DNA was extracted from T-I cells attached to the culture dishes as described previously (Tilly and Hsueh, 1993). Briefly, cells were disrupted with homogenization buffer on ice and the homogenates were then lysed. The DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1; v:v:v). The quantity and purity of each sample were measured spectrophotometrically at 260 and 280 nm absorbance.
Autoradiographic analysis of internucleosomal DNA cleavage
Apoptosis was identified by detection of internucleosomal cleavage of LMW DNA as described by Tilly and Hsueh (1993). The quantity of each DNA sample was determined spectrophotometrically immediately after isolation and before labeling. Aliquots of 250 ng of DNA from each sample were labelled on 3'-ends with [32P] dideoxy-ATP (10 mCi/ml) using the terminal transferase (25 U) reaction. Labelled samples (125 ng/lane) were separated by electrophoresis through a 2% agarose gel, dried in a slab-gel dryer without heat and exposed to a film at –70°C for up to 24 h. Following auto radiographic exposure, portions of the gel corresponding to the LMW DNA fraction (<10 kb) of each sample were excised and the amount of [32P] incorporated into the DNA was determined using a ß-counter SL 4000 (Intertechnique, Fairfield, NJ, USA). The amount of radiolabelled ddATP incorporated into LMW DNA fractions was used to evaluate the proportion of internucleosomal DNA fragmentation in all samples.
Statistical analysis
Results are presented as the mean ± SEM of four experiments, unless stated otherwise. Comparisons between the means were performed using a non-parametric approach with Kruskal–Wallis analysis of variance by ranks followed by pairwise comparisons using the Mann–Whitney U test. Results were considered to be significantly different at P<0.05.
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Results |
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Histological assessment of nuclear morphology of rat T-I cells cultured without serum showed characteristic features of apoptotic cell death, such as nuclear condensation and fragmentation into apoptotic bodies, in a proportion of T-I cells (Figure 1). Morphologic appearance of apoptotic cells was consistent with mesenchymal cells. Apoptotic cells were almost exclusively found in serum deprived T-I cultures as compared to T-I cultures supplemented with 10% FBS. Asynchronicity of apoptosis and its duration of several hours result in small numbers of apoptotic cells at a given time point and make detection of apoptotic cell death by morphology difficult. Hence, the sensitive nature of 3' end-radiolabelling of DNA allowed for qualitative and quantitative determination of differences in apoptosis between distinct treatments.
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The time course of the effect of serum withdrawal on LMW DNA fragmentation in T-I cells is presented in Figure 2. The cells were cultured for 24 h with 10% FBS, washed and then incubated with or without 10% FBS for 6, 12 and 24 h. In the presence of serum, the internucleosomal DNA cleavage was minimal at all incubation periods. Radiolabelled DNA extracted from serum-deprived T-I cells produced characteristic DNA ladders at all time-points tested; the most extensive LMW DNA fragmentation was observed after 24 h of serum deprivation. IGF-I (10 nM) suppressed apoptotic DNA breakdown at all studied time intervals.
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The comparison of relative anti-apoptotic potencies of insulin, IGF-I, or IGF-II is presented in Figure 3. T-I cells were cultured for 24 h with 10% FBS, washed and incubated for an additional 24 h without serum or with 10% FBS, insulin, IGF-I, or IGF-II. LMW DNA fragmentation was decreased in the presence of serum by 80 ± 3% in comparison to serum-free controls (P<0.005, n=4). In serum-free cultures supplemented with insulin, the LMW DNA labeling was decreased by 39 ± 8% versus serum-free controls (P=0.002, n=4). A comparable reduction in apoptotic DNA fragmentation was achieved in cultures of T-I cells in serum-free media supplemented with IGF-I or IGF-II as reflected by the decreases of the LMW DNA labelling of 55 ± 9% (P<0.05, n=4) and 37 ± 14% (P=0.02, n=4), respectively.
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The role of LH in regulating T-I cell apoptosis was evaluated by determination of 3'-end labeling of LMW DNA fragments after 24 h culture of T-I cells with 10% FBS followed by another 24 h incubation with LH or the cyclic AMP analog, 8Br-cAMP, in the absence of FBS (Figure 4). Serum withdrawal induced DNA fragmentation and LH augmented this effect, increasing the incorporation of radiolabelled ddATP by 85 ± 55% above serum-free controls (P=0.03, n=4). 8Br-cAMP caused a similar potentiation of apoptosis, increasing LMW DNA cleavage by 72 ± 42% in comparison to serum-free controls (P=0.03, n=4).
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Discussion |
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The present study, using an in vitro model, demonstrates that apoptosis of purified rat ovarian T-I cells is regulated by endocrine factors such as insulin, insulin-like growth factors and LH. We have shown that serum withdrawal is a potent inducer of T-I cell apoptosis, identified by characteristic morphological features (nuclear condensation and apoptotic bodies) and internucleosomal DNA cleavage detected by 3' end-labeling. This apoptotic effect of serum deprivation is partly reversed by insulin, IGF-I or IGF-II, all of which had comparable potencies in their suppressive actions on LMW DNA fragmentation. In contrast, LH enhanced apoptosis of cultured T-I cells, augmenting internucleosomal DNA cleavage caused by serum deprivation. Moreover, this action was likely mediated by stimulation of the cAMP system.
At present, mechanisms regulating apoptosis of T-I cells are still poorly understood. In situ studies revealed that the predominant site of apoptosis in the ovary is the granulosa cell compartment (Palumbo and Yeh, 1994; Tilly et al., 1995; Kugu et al., 1998) but apoptotic DNA fragmentation was also shown in a small number of theca cells of atretic rat follicles (Palumbo and Yeh, 1994). In contrast, Billig et al. detected no apoptotic DNA breakdown in ovarian theca and interstitial cells in a rat model using estrogen withdrawal (Billig et al., 1993). The proportion of T-I cells undergoing spontaneous apoptosis is very small as demonstrated by our studies with in situ 3' end-labeling (TUNEL technique) of cultured T-I cells and rat ovarian sections (results not shown). This finding is confirmed by the current observation of a relatively limited fraction of cultured T-I cells that express the morphological features of apoptotic death. The difficulty in detecting apoptosis among T-I cells may be due to the nature of this process. T-I cells do not undergo extensive spontaneous waves of apoptosis so characteristic among granulosa cells during follicular atresia. Most likely death of cells within the T-I compartment is not a rapid synchronized process, rather it occurs in an ongoing fashion over an extended period of time. Indeed, there is evidence suggesting that a slow process of T-I apoptosis takes place at final stages of follicular atresia (Logothetopoulos et al., 1995).
In the present study, the serum withdrawal model of apoptosis was used. Such model has been widely used to study apoptosis in cultures of various cells including adipocytes (Valverde et al., 2004), malignant myeloid cells (Valverde et al., 2004) and neurons (Benedito et al., 2004). However, it is important to acknowledge such an in vitro model that represents a departure from physiological conditions and therefore, the findings should be interpreted with caution.
Our in vitro model, with DNA extraction and analysis of internucleosomal DNA cleavage, does not allow us to discriminate which subtypes of T-I cells undergo apoptosis. It is reported that during follicular atresia some T-I cells are eradicated by the apoptotic process, but some differentiate into ovarian interstitium, retain LH receptors and become steroidogenically active, producing predominantly progesterone (Erickson et al., 1985; Palumbo and Yeh, 1994). While DNA laddering does not directly reflect the number and types of cells undergoing apoptosis, it is probably one of the most sensitive, quantitative methods allowing the analysis of internucleosomal DNA fragmentation in cultured cells exposed to different treatments.
Apoptosis is essential in cell development and tissue remodelling. Cells undergo apoptotic death through the activation of specific apoptotic pathways and/or through the inactivation of survival mechanisms (Igney and Krammer, 2002). It seems reasonable to propose that the regulation of T-I survival versus death should be a major factor in the maintenance of ovarian homeostasis. Control of the balance between cell proliferation and death ensures appropriate follicular development and normal size of the T-I ovarian compartment. The fate of the T-I cell is determined by signals from different endocrine and paracrine regulators such as growth factors and gonadotrophins. Insulin, IGF-I, IGF-II and their receptors have been identified in the ovary and were shown to modulate ovarian steroidogenesis (el-Roeiy et al., 1993; Cara, 1994). A family of IGF-binding proteins (IGFBPs) and their proteases are also produced by rat ovarian cells and may affect the actions of IGFs, most likely by modulating their bioavailability. T-I cells predominantly express and produce IGFBP-2, 3, 6, with corresponding proteases and their interplay could determine the net bioavailability of IGF peptides (Nakatani et al., 1991; Poretsky et al., 1999). We have previously demonstrated that insulin, IGF-I and IGF-II dose-dependently stimulate, predominantly by type I-IGF receptors, DNA synthesis of T-I cells with a resultant increase in the number of steroidogenically active cells (Duleba et al., 1997). The present findings offer a new potential explanation for the above observations. An increase in the total T-I cell number may be not only due to the proliferative effects of insulin and IGFs but also be a result of their antiapoptotic action. Insulin and IGFs may act as survival factors to suppress apoptosis in T-I cells and hence act to increase the pool of viable cells, some of which undergo proliferation. Thus, these agents may participate in the regulation of the size of T-I compartment by affecting both cell growth and death. Insulin and IGF-I were previously shown to suppress apoptosis in cultured early antral and preovulatory follicles of the rat as well as in cultured porcine granulosa cells (Chun et al., 1994, 1996; Guthire et al., 1997). T-I cells in our model could produce or release selected IGFBPs in vitro and consequently decrease the antiapoptotic potency of IGFs. Additionally, activity of IGFBP proteases could modulate the levels of IGFBPs in culture and hence influence the intensity of apoptosis. As demonstrated in other tissues, the action of these survival factors may be mediated through the activation of PI-3 and MAPK kinases (Parrizas et al., 1997). Additionally insulin and IGFs may also upregulate-selected inhibitors of apoptosis proteins that were recently localized in the ovary (Asselin et al., 2001; Chun et al., 2001; Johnson et al., 2002). The present report extends previous observations and demonstrates the antiapoptotic effect of insulin and both IGFs in isolated T-I cells.
Another important observation in this study is the demonstration that LH or a cAMP analogue augments the apoptotic effect of serum withdrawal on T-I cells. This complements our previous findings that LH and cAMP analogues inhibit DNA synthesis and decreases the total number of cultured T-I cells (Duleba et al., 1999). Therefore, we propose that LH, acting via the cAMP system, affects T-I DNA synthesis and cell number by increasing the rate of T-I cell apoptosis rather than by any direct effect on proliferation. Hence, despite its well-known stimulatory action on T-I androgen production, LH may have a counter-regulatory effect to oppose the actions of insulin and IGFs on the size of the T-I compartment. LH activates several different signal transduction pathways; however, in our system, activation of the cAMP pathway seems to be most plausible. Interestingly, cAMP was shown to activate apoptosis in rat and human granulosa cells by upregulation of death promoters (p53 and bax) with no alteration in death repressor genes (Zwain and Amato, 2001).
Interestingly, similar observation of LH-induced apoptosis has been demonstrated in the T-I cells of rat preovulatory follicles in culture (Yacobi et al., 2004). Surprisingly, addition of a presumably survival factor—LH, increased caspase-3 activity that localized predominantly to T-I cells. Elevated caspase-3 activity was accompanied by increase in T-I cell apoptosis as demonstrated by TUNEL staining and DNA fragmentation analysis. Despite, the documented role of LH in thecal hypertrophy during late stages of follicular atresia, this gonadotrophin may have dual action and may exert proapoptotic actions during early follicular growth.
The present findings may also have some clinical relevance. Hyperplasia and dysfunction of the T-I cell compartment are characteristic of conditions such as, PCOS and hyperthecosis (Hughesdon, 1982). PCOS is also associated with insulin resistance, compensatory hyperinsulinemia and increased levels of free IGF-I (Dunaif et al., 1989; Homburg et al., 1992). We speculate that in women with PCOS, excessive levels of insulin and free IGF-I or IGF-II may limit apoptotic death of T-I cells and thus lead to thecal and stromal hyperplasia. Testing of this hypothesis will require further studies, including determination whether our present observations are also applicable to T-I cells from other species, especially human.
In summary, the present report demonstrates that programmed cell death of rat ovarian T-I cells is regulated by insulin, IGFs and LH. Further studies will focus on elucidating the intracellular mechanisms transducing survival/apoptosis signals.
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Acknowledgements |
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We would like to acknowledge that ovine LH (o-LH-26) was generously donated by the National Hormone and Pituitary Program, NIDDK and Dr A.F. Parlow. The study was supported by NIH grant R01 HD40207 (to AJD). Investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Asselin E, Wang Y and Tsang B (2001) X-linked inhibitor of apoptosis protein activates the phosphatidylinositol 3-kinase/Akt pathway in rat granulosa cells during follicular development. Endocrinology 142, 2451–2457.
Benedito A, Lehtinen M, Massol R, Kirchhausen T, Rao A and Bonni A (2004) The transcription factor NFAT3 mediates neuronal survival. J Biol Chem 280, 2818–2825.
Billig H, Furuta I and Hsueh AJ (1993) Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology 133, 2204–2212.[Abstract]
Cara JF (1994) Insulin-like growth factors, insulin-like growth factor binding proteins and ovarian androgen production. Horm Res 42, 49–54.[ISI][Medline]
Cara JF and Rosenfield RL (1988) Insulin-like growth factor I and insulin potentiate luteinizing hormone-induced androgen synthesis by rat ovarian thecal-interstitial cells. Endocrinology 123, 733–739.[Abstract]
Caubo B, DeVinna RS and Tonetta SA (1989) Regulation of steroidogenesis in cultured porcine theca cells by growth factors. Endocrinology 125, 321–326.[Abstract]
Chun S, Bae H, Kim W, Park J, Hsu S and Hsueh A (2001) Expression of messenger ribonucleic acid for the antiapoptosis gene P11 in the rat ovary: gonadotrophin stimulation in granulosa cells of preovulatory follicles. Endocrinology 142, 2311–2317.
Chun SY, Billig H, Tilly JL, Furuta I, Tsafriri A and Hsueh AJ (1994) Gonadotrophin suppression of apoptosis in cultured preovulatory follicles: mediatory role of endogenous insulin-like growth factor I. Endocrinology 135, 1845–1853.[Abstract]
Chun SY, Eisenhauer KM, Minami S, Billig H, Perlas E and Hsueh AJ (1996) Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor. Endocrinology 137, 1447–1456.[Abstract]
Duleba AJ, Spaczynski RZ, Olive DL and Behrman HR (1997) Effects of insulin and insulin-like growth factors on proliferation of rat ovarian theca-interstitial cells. Biol Reprod 56, 891–897.[Abstract]
Duleba AJ, Spaczynski RZ, Olive DL and Behrman HR (1999) Divergent mechanisms regulate proliferation/survival and steroidogenesis of theca-interstitial cells. Mol Hum Reprod 5, 193–198.
Dunaif A, Segal KR, Futterweit W and Dobrjansky A (1989) Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes 38, 1165–1174.[Abstract]
Erickson GF, Magoffin DA, Dyer CA and Hofeditz C (1985) The ovarian androgen producing cells: a review of structure/function relationship. Endor Rev 6, 371–399.[ISI][Medline]
el-Roeiy A, Chen X, Roberts VJ, LeRoith D, Roberts CT, Jr and Yen SS (1993) Expression of insulin-like growth factor-I (IGF-I) and IGF-II and the IGF-I, IGF-II and insulin receptor genes and localization of the gene products in the human ovary. J Clin Endocrinol Metab 77, 1411–1418.[Abstract]
Foghi A, Teerds KJ, van der Donk H and Dorrington J (1997) Induction of apoptosis in rat thecal/interstitial cells by transforming growth factor alpha plus transforming growth factor beta in vitro. J Endocrinol 153, 169–178.[Abstract]
Foghi A, Ravandi A, Teerds KJ, Van Der Donk H, Kuksis A and Dorrington J (1998) Fas-induced apoptosis in rat thecal/interstitial cells signals through sphingomyelin-ceramide pathway. Endocrinology 139, 2041–2047.
Guthire H, Garrett W and Cooper B (1997) Follicle-stimulating hormone and insulin-like growth factor-I attenuate apoptosis in cultured porcine granulosa cells. Biol Reprod 58, 390–396.
Homburg R, Pariente C, Lunenfeld B and Jacobs HS (1992) The role of insulin-like growth factor-1 (IGF-1) and IGF binding protein-1 (IGFBP-1) in the pathogenesis of polycystic ovary syndrome. Hum Reprod 7, 1379–1383.
Hughesdon PE (1982) Morphology and morphogenesis of the Stein-Leventhal ovary and of so called ‘hyperthecosis’. Obstet Gynecol Surv 37, 59–77.[Medline]
Hughes FM and Gorospe WC (1991) Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia. Endocrinology 129, 2415–2422.[Abstract]
Huppertz B, Frank H-G and Kaufmann P (1999) The apoptosis cascade—morphological and immunohistochemical methods for its visualization. Anat Embryol 200, 1–18.[CrossRef][Medline]
Igney F and Krammer P (2002) Death and anti-death: tumor resistance to apoptosis. Nature Rev Cancer 2, 277–288.[CrossRef][ISI][Medline]
Johnson A, Langer J and Bridgham J (2002) Survivin as a cell cycle-related and antiapoptotic protein in granulosa cells. Endocrinology 143, 3405–3413.
Kaipia A and Hsueh AJ (1997) Regulation of ovarian follicle atresia. Ann Rev Physiol 59, 349–363.[CrossRef][ISI][Medline]
Kondo H, Maruo T, Peng X and Mochizuki M (1996) Immunological evidence for the expression of the Fas antigen in the infant and adult human ovary during follicular regression and atresia. J Clin Endocrinol Metab 81, 2702–2710.[Abstract]
Kugu K, Ratts VS, Piquette GN, Tilly KI, Tao XJ, Martimbeau S, Aberdeen GW, Krajewski S, Reed JC, Pepe GJ et al. (1998) Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary. Cell Death Differ 5, 67–76.[CrossRef][ISI][Medline]
Logothetopoulos J, Dorrington J, Bailey D and Stratis M (1995) Dynamics of follicular growth and atresia of large follicles during the ovarian cycle of the guinea pig: fate of the degenerating follicles, a quantitative study. Anat Rec 243, 37–48.[CrossRef][Medline]
Magoffin DA and Erickson GF (1988) Purification of ovarian theca-interstitial cells by density gradient centrifugation. Endocrinology 122, 2345–2347.[Abstract]
Nakatani A, Shimasaki S, Erickson G and Ling N (1991) Tissue-specific expression of four insulin-like growth factor-binding proteins (12, 3 and 4) in the rat ovary. Endocrinology 129, 1521–1529.[Abstract]
Palumbo A and Yeh J (1994) In situ localization of apoptosis in the rat ovary during follicular atresia. Biol Reprod 51, 888–895.[Abstract]
Parrizas M, Saltiel A and LeRoith D (1997) Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3'-kinase and mitogen activated protein kinase pathways. J Biol Chem 272, 154–161.
Pehlivan T, Mansour A, Spaczynski RZ and Duleba A (2001) Effects of transforming growth factors-alpha and -beta on proliferation and apoptosis of rat theca-interstitial cells. J Endocrinol 170, 639–645.[Abstract]
Poretsky L, Cataldo N, Rosenwaks Z and Giudice L (1999) The insulin-related ovarian regulatory system in health and disease. Endocr Rev 20, 535–582.
Porter D, Vickers S, Cowan R, Huber S and Quirk S (2000) Expression and function of Fas antigen vary in bovine granulosa and theca cells during ovarian follicular development and atresia. Biol Reprod 62, 62–66.
Stadelmann C and Lassmann H (2000) Detection of apoptosis in tissue sections. Cell Tissue Res 301, 19–31.[CrossRef][ISI][Medline]
Tilly JL and Hsueh AJ (1993) Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation. J Cell Physiol 154, 519–526.[CrossRef][ISI][Medline]
Tilly JL, Kowalski KI, Johnson AL and Hsueh AJ (1991) Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 129, 2799–2801.[Abstract]
Tilly JL, Kowalski KI, Schomberg DW and Hsueh AJ (1992) Apoptosis in atretic ovarian follicles is associated with selective decreases in messenger ribonucleic acid transcripts for gonadotrophin receptors and cytochrome P450 aromatase. Endocrinology 131, 1670–1676.[Abstract]
Tilly JL and Robles R (1999) In Fauser BCMJ, Rutheford AJ, Strauss JF, III, and van Steirteghem A (eds) Molecular Biology in Reproductive Medicine. Parthenon, New York, pp. 79–101.
Tilly JL, Tilly KI, Kenton ML and Johnson AL (1995) Expression of members of the bcl-2 gene family in the immature rat ovary: equine chorionic gonadotrophin-mediated inhibition of granulosa cell apoptosis is associated with decreased bax and constitutive bcl-2 and bcl-xlong messenger ribonucleic acid levels. Endocrinology 136, 232–241.[Abstract]
Valverde AM, Mur C, Brownlee M and Benito M (2004) Susceptibility to apoptosis in insulin-like growth factor-I receptor-deficient brown adipocytes. Mol Biol Cell 15, 5101–5117.
Yacobi K, Wojtowicz A, Tsafriri A and Gross A (2004) Gonadotrophins enhance caspase-3 and -7 activity and apoptosis in the theca-interstitial cells of rat preovulatory follicles in culture. Endocrinology 145, 1943–1951.
Zerbinatti C, Mayer L, Audet R and Dyer C (2001) Apolipoprotein E is a putative autocrine regulator of the rat ovarian theca cell compartment. Biol Reprod 64, 1080–1089.
Zwain I and Amato P (2001) cAMP-induced apoptosis in granulosa cells is associated with up-regulation of P53 and bax and down-regulation of clusterin. Endocr Res 27, 233–249.[CrossRef][ISI][Medline]
Submitted on January 31, 2005; accepted on March 14, 2005.
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