Effects of dehydroepiandrosterone on ovarian cystogenesis and immune function

Journal of Reproductive Immunology 64 (2004) 59–74

Effects of dehydroepiandrosterone on ovarian cystogenesis and immune function Carolina Griselda Luchetti a , Maria Emilia Solano a , Valeria Sander a , Maria L. Barreiro Arcos a , Claudio Gonzalez b , Guillermo Di Girolamo b , Sara Chiocchio c , Graciela Cremaschi a , Alicia B. Motta a,∗ a

Laboratorio de Fisiopatolog´ıa Ovárica, Centro de Estudios Farmacológicos y Botánicos (CEFYBO), Consejo Nacional de Investigaciones Cient´ıficas y Técnicas (CONICET), Serrano 669, C1414DEM Buenos Aires, Argentina b Departamento de Farmacolog´ıa, Facultad de Medicina, Buenos Aires, Argentina c Instituto de Neurobiolog´ıa (IDNEU), Buenos Aires, Argentina Received in revised form 22 April 2004; accepted 23 April 2004

Abstract The purpose of the present report was to study the possible relationship between ovarian functionality and the immune response during cystogenesis induced by androgenization with dehydroepiandrosterone (DHEA). Daily injection of DHEA (6 mg/kg body weight) for 20 consecutive days induced ovarian cysts in BALB/c mice. As markers of ovarian function, serum estradiol (E) and progesterone (P) and the ovarian inmunomodulator prostaglandin E (PGE) were analyzed. In order to know how the integrity of the tissue was altered after induction of cystogenesis, the oxidative status was also evaluated. Serum E and P levels, and ovarian PGE concentration, were increased in animals with cysts compared with healthy controls. The oxidant status (quantified by malondialdehyde (MDA) formed after the breakdown of the cellular membrane by free radical mechanisms) was augmented, meanwhile the antioxidant (evaluated by the glutathione (GSH) content) diminished during the induction of cystogenesis. Both immunohistochemical and flow cytometry assays demonstrated that DHEA treatment increased the number of T lymphocytes infiltrating ovarian tissue. Therefore, while ovarian controls showed equivalent expression of CD4+ and CD8+ T cell subsets, injection of DHEA yielded a selective ovarian T cell infiltration as demonstrated by enhanced CD8+ and diminished CD4+ T lymphocyte expression. These results show that the development of cysts involves changes in ovarian

∗

Corresponding author. Fax: +54 11 48562751. E-mail address: [email protected] (A.B. Motta).

0165-0378/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2004.04.002

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function and an imbalance in the oxidant–antioxidant equilibrium. We observed also both an increased and selective T lymphocyte infiltration. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Dehydroepiandrosterone; Lymphocyte; Polycystic ovary syndrome; CD4+ and CD8+; Estrogen; Progesterone; Oxidative parameters

1. Introduction The current diagnostic criteria for polycystic ovary syndrome (PCOS) is hyperandrogenism and ovulatory dysfunction with the exclusion of specific disorders such as congenital adrenal hyperplasia. Polycystic ovaries display increased ovarian stroma and subcapsular follicular cysts (Franks, 1995). It has long been recognized as a heterogeneous disease that can result in hypersecretion of circulating LH but with lower or equivalent FSH levels, follicular atresia, insulin resistance and infertility (Abbott et al., 2002). Despite its prevalence, little is known about the aetiology and pathophysiology of the syndrome. However, during the last decade, several clues have emerged from human and animal studies that may have significant repercussions in the treatment. The battery of animal models used for the study of polycystic ovaries (Billiar et al., 1985; Szkiewicz and Uilenbroek, 1998; Weil et al., 1999; West et al., 2001; Abbott et al., 2002) have allowed a focus on different aspects of the pathology. Mahesh and Greenblatt (1962) were among the first to isolate dehydroepiandrosterone (DHEA) from the ovarian tissue of women with PCOS. After that, Roy et al. (1962) produced an animal model for the study of PCOS using DHEA. Subsequent studies confirmed that the DHEA-PCOS murine model exhibits many of the salient features of human PCOS (Lee et al., 1991; Anderson et al., 1992). These findings coupled to the fact that increased evidence indicates that DHEA has in addition, potent immunoregulatory functions, such as enhancing the ability of mice to resist experimental and bacterial diseases (Araghi-Niknam et al., 1997; Hernandez-Pando et al., 1998; Carr, 1998; Zhang et al., 1999; Ben et al., 1999; Du et al., 2001) and many human chronic inflammatory pathologies (Vollenhoven et al., 1994), emphasize the need for more information concerning the possible relationship between DHEA and the immune response in ovarian cystogenesis. Thus, the present experiments were designed to study the possible role of DHEA in the immune response in cystogenesis induced in mice. In order to determine how the functionality of ovarian tissue was modified with cystogenesis, the endocrine markers serum progesterone (P) and estradiol (E), as well as ovarian prostaglandin E (PGE), were evaluated. It is important to note that PGs modulate different ovarian functions, such as the rupture of ovarian follicles associated with ovulation (Husein and Kridli, 2003; Medan et al., 2003) and luteolysis (Motta et al., 1999, 2001b). Moreover, increased levels of circulating PGE2 support up-regulation of T lymphocytes during excessive exercise (Lakier, 2003), mediate an immune response with a suppressive role in T lymphocytes in BALB/c mice (Kuroda and Yamashita, 2003) and are associated with tumors by means of suppressing differentiation of dendritic cells (Yang et al., 2003). Particularly, PGE has been studied as an immunosuppressive molecule (Wojtowicz-Praga, 1997). Moreover, this prostanoid has been found to be augmented in patients with PCOS (Navarra et al., 1996). In the present report ovarian

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PGE production was evaluated both as a measurement of the inflammatory process and by its immunomodulatory property. Finally, considering the important damage that ovarian tissue suffers in cyst pathology, we examined the possible imbalance in oxidant–antioxidant status by quantifying lipid peroxidation (as a measurement of the oxidative condition) and glutathione content (which serves as endogenous antioxidant) in ovaries from mice with induced cystogenesis in comparison with healthy controls. It is relevant to point out that the association between oxidant and antioxidant status has been reported not only in other pathologies such as aging (Niwa et al., 1993), diabetes mellitus (Vural et al., 2001), infectious diseases (Erel et al., 1997), cerebral disease (Finotti et al., 2000) and as marker of the cardiovascular risk factor (Sabuncu et al., 2001) but also recently it was related with the polycystic kidney disease (Maser et al., 2002). 2. Materials and methods 2.1. Animals and experimental protocol In order to study the effects of high levels of circulating androgens, we used a dehydroepiandrosterone (DHEA) mouse model able to induce cystogenesis (Lee et al., 1991). Briefly, female prepuberal (25 days old) mice of the BALB/c strain were injected (sc) daily with DHEA (6 mg/kg body weight) dissolved in 0.1 ml sesame oil. First, animals were treated during different consecutive days to analyze the apparition of cysts. As it was observed that mice developed ovarian cysts after 20 consecutive days of DHEA treatment, this was the time chosen to carry out the experiments. Mice (40 per group: control and DHEA-treated) were housed under controlled temperature (22 ◦ C) and illumination (14-h light, 10-h darkness; lights on at 05:00 h) and were allowed free access to Purina rat chow and water. All procedures involving animals were approved by the Animal Care and Use Committee of the National Council Research (CONICET). Throughout the whole treatment, sexual cycle was determined by daily vaginal smears. Considering that DHEA-treated animals exhibited constant estrus, while animals injected with oil showed different stages of the cycle, the control group was collected from animals injected with 0.1 ml of oil during 20 consecutive days at the estrus stage. After 20 days of DHEA treatment, mice were anesthetized with ether and sacrificed by decapitation. Blood was collected for hormone analysis (P and E) and freshly dissected ovaries were divided as following: 10 of each group (control or DHEA) were immediately fixed in 4% (w/v) paraformaldehyde for morphological and immunohistochemical studies while the remaining tissues were immediately frozen at −20 ◦ C until used (from each group: 10 ovaries were employed for PGE determination, 30 for glutathione and 30 for lipid peroxidation assays). For flow cytometry assay, ovaries were obtained from animals of control (10 mice) and DHEA-treated (10 mice) groups injected as described above. All experiments were repeated three times. 2.2. Morphological studies To study the effect of DHEA on cyst formation, five ovaries from the control group and five from DHEA-treated, fixed as described above, were consecutively cut (6 ␮m per section)

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and placed on gelatin-coated slides (Biobond, British Biocell International, Cardiff) and air-dried for 2 h before being fixed for 5 min in acetone at 4 ◦ C. Then, consecutive sections of each ovary were washed in PBS (pH 7.3) and stained with haematoxylin and eosin (DAKO Corporation, Carpinteria, CA, USA) for histological analysis of cyst formation. This resulted in forty sections for each ovary. For morphological analysis, the sections were chosen as following: five of each extreme and five of the middle of each ovary. Five ovaries from the control and five from the treated group were observed. 2.3. Estradiol and progesterone determination To evaluate ovarian function after cystogenesis induction, P and E were measured in serum samples as described before (Motta et al., 2001b). Briefly, blood was allowed to clot and serum removed and frozen until progesterone or estradiol concentrations were determined by radioimmunoassay. Both antisera were provided by Dr. G.D. Niswender (Colorado State University, Fort Collins, CO, USA). The progesterone antiserum was highly specific for progesterone with low cross-reactivity <2% for 20 ␣-dihydro-progesterone and deoxycorticosterone, and 1% for other steroids normally present in the serum. The sensitivity was 5–10 pg/tube, so 2–5 ␮l of serum was routinely assayed. The estradiol antiserum showed low cross-reactivity <1% for progesterone and testosterone, <5% for 17-estradiol and estriol and <10% for estrone. Results were expressed as ng/ml serum. 2.4. Prostaglandin radioimmunoassay The measurement of PGE was carried out in incubation media, since previously we had determined that both homogenates and incubated tissue reflected ovarian PGE levels. Briefly, the tissue (each ovary) was weighed and incubated in Krebs–Ringer-bicarbonate (KRB) with glucose (11.0 mmol/l) as external substrate (pH 7.0) for 1 h in a Dubnoff metabolic shaker under an atmosphere of 5% CO2 in 95% O2 at 37 ◦ C. At the end of the incubation period, the tissue was removed and the solution acidified to pH 3.0 with 1 M HCl and extracted for prostaglandin determination three times with 1 volume of ethyl acetate. Pooled ethyl acetate extracts were dried under an atmosphere of N2 and stored at −20 ◦ C until prostaglandin radioimmunoassay was performed. Prostaglandin E was quantified using a rabbit antiserum from Sigma Chemical Co. (St. Louis, MO, USA). Sensitivity was 10 pg/tube and cross-reactivity was 100% PGE and <0.1% with other prostaglandins. Results were expressed as pg PGE/mg protein. Ovarian protein content was determined by Bradford method. 2.5. Oxidative stress-related parameters 2.5.1. Glutathione content (GSH) The glutathione assay was carried out as previously described (Motta et al., 2001a). The reduced form of glutathione (GSH) comprises the bulk of cellular protein sulphydryl groups. Thus, measurement of acid-soluble thiol is commonly used for estimation of GSH content in tissue extracts. Briefly, 300 ␮l of homogenates obtained from pooled tissues (three ovaries from different animals/point) in 0.5% (v/v) trichloroacetic acid were incubated with

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buffer—1.75 M Tris (pH 7.4) containing NADPH and glutathione reductase. The reaction involves enzymatic reduction of the oxidized form (GSSG) to GSH. When Ellman’s reagent (a sulphydryl reagent, 5,5-dithiobis-2 nitrobenzoic acid; Sigma Chemical Co., USA) is added to the incubation medium, the chromophoric product resulting from this reaction develops a molar absorption at 412 nm that is linear up to 6 min; after this, the reaction remains constant. Results were expressed as ␮mol GSH/g ovarian tissue. 2.5.2. Lipid peroxidation The amount of malonialdehyde formed from the breakdown of polyunsatured fatty acids may be taken as an index of peroxidation reaction. The method used in the present study was as previously described (Motta et al., 2001a) and quantifies malondialdehyde as the product of lipid peroxidation that reacts with trichloracetic acid–thiobarbituric acid–HCl (15% (w/v); 0.375% (w/v) and 0.25 M, respectively) yielding a red compound that absorbs at 535 nm. Homogenates of ovarian pooled tissue (three ovaries/point) were treated with trichloracetic acid–thiobarbituric acid–HCl and heated for 15 min in boiling water bath. After cooling, the flocculent precipitate was removed by centrifugation at 1000 g for 10 min. The absorbance of samples was determined at 535 nm. Content of thiobarbituric acid reactants were expressed as ␮mol malondialdehyde (MDA) formed/g ovarian tissue. 2.6. Immunohistochemistry The presence and percentages of CD4+ T-helper/inducer and CD8+ T-cytotoxic/ suppressor lymphocytes were evaluated by immunohistochemical staining of ovarian sections with fluorescein (FITC)-conjugated anti-mouse-CD8+ or with phycoerythrin (PE)conjugated anti-mouse-CD4+ T cell monoclonal antibodies (Sigma Chemical Co.). Briefly, cryostat sections (6 ␮m) were cut consecutively for each sample, placed on gelatin-coated (Biobond, British Biocell International, Cardiff) slides and air-dried for 12 h before they were fixed for 5 min with acetone at 4 ◦ C. Sections were washed with 0.01 M PBS (pH 7.3) and treated with 1 ␮g (4 ␮l) of the respective monoclonal antibody. Incubation was carried out in darkness at room temperature overnight. Percentages of CD4+ and CD8+ T cells were determined by fluorescence microscopy. Isotype controls (IgG1-FITC and IgG2a-PE) were used in paired samples to determine non-specific staining. Cell suspensions obtained from BALB/c lymph nodes were used as positive controls. Five ovaries from control and five from DHEA-treated animals were analyzed. 2.7. Flow cytometry To carry out the flow cytometry assay, ovarian cells must be dispersed. Briefly, ovaries (from control and DHEA-treated group, 10 animals for each one) were enzymatically dissociated in culture medium (medium 199, 25 mM NaHCO3 , 26 mM Hepes, and 50 IU/ml penicillin) with trypsin-free collagenase (740 IU/100 mg tissue) and DNAse (14 IU/100 mg tissue). After 90 min, ovarian cells were washed twice with culture medium, twice with Dulbeco-phosphate-buffered saline free of Ca++ and Mg++ (PBS), and twice with culture medium containing EDTA (1 mM). To remove blood cells, suspensions were applied to a

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Ficoll-hystopaque gradient 1.077 (Sigma, St. Louis, USA), centrifuged at 400 g for 45 min and washed with PBS/0.1% BSA. Cells were counted in a haemocytometer, the viability >80% as assessed by the trypan blue exclusion method, and then processed by direct immunofluorescence. Thus, 100 ␮l of each cellular suspension (control or DHEA-treated), at a concentration of 106 cells/ml, was incubated during 30 min at 4 ◦ C with: (i) 30 ␮l of phycoerythrin (PE) rat IgG2a␬ isotype control plus 30 ␮l of fluorescein (FITC) rat IgG2a␬ isotype control (eBioscience, USA) corresponding the isotype control sample or (ii) 4 ␮l (=8 ug) PE anti-mouse-CD4 plus 4 ␮l (=8 ug) FITC anti-mouse-CD8 T cell monoclonal antibody (eBioscience, USA) corresponding to control or DHEA assay according to the cellular suspension. Antibodies were used at saturating concentration as established after titration by flow cytometry. Then, samples were washed with PBS and PBS–EDTA, fixed with 4% paraformaldehyde and stored at 4 ◦ C in darkness until analysis within 6 days of labeling. Fluorescence analysis was evaluated with FACScan® and the Winmdi 2.8 software. Lymphocytes were analyzed using different physical characteristics (that is, size and complexity) by gating using forward (FSC: cell size) and side scatter (SSC: cell complexity) parameters. Flow cytometric analysis was performed using standard fluorescence 1 (FL1: FITC anti-mouse-CD8+ T lymphocytes) and fluorescence 2 (FL2: PE anti-mouse-CD4+ T lymphocytes). The analysis was based on measurement of 50,000 nucleated cells/assay where by means of the size and complexity, the specific region of the T lymphocyte was characterized. The percentage of positively labeled cells was calculated by subtracting signals from non-specifically labeled cells. 2.8. Statistical analysis Statistical analyses were carried out using the Instat program (GraphPAD software, San Diego, CA, USA). Student t-test was used for comparisons between value of groups; P < 0.05 was considered significant.

3. Results 3.1. Morphology Fig. 1 A shows a representative ovarian tissue section from the control group. The general appearance of the tissue resembles normal histology: a central medulla consisting of a fibromuscular stroma and a large number of blood vessels and a peripheral cortex containing large numbers of follicles in various stages of development. Histological examination of ovaries from DHEA-treated mice revealed an increase in fat and stroma tissues and enhanced leukocyte infiltration (Fig. 1B). The cortex showed an increase in the number of atretic follicles. Treatment also yielded formation of cysts (no more than two for each ovary). The morphology of cysts is characterized by a thin layer of theca cells and a compacted formation of granulosa cells; the vascularized theca interna is absent in cysts (Fig. 1C).

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Fig. 1. (A) A representative ovarian tissue section of mouse injected with oil for 20 consecutive days on estrus stage (×100); (B) an ovarian tissue section from DHEA-treated mouse for 20 consecutive days (×100); (C) detail of ovarian cyst.

3.2. Ovarian function: serum estradiol and progesterone levels, ovarian PGE production As a measurement of ovarian function in the cyst pathology, serum E and P levels and ovarian PGE production were evaluated by specific radioimmunoassays. DHEA treatment

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increased both E and P serum levels (Fig. 2A and B, respectively). The ovarian immunosuppresor PGE was also increased after DHEA treatment (Fig. 2C) compared with controls. 3.3. Glutathione levels and lipid peroxidation index To evaluate the oxidative status during development of the ovarian cystogenesis, total GSH (both reduced and oxidized forms) and lipid peroxidation index were measured. Con***

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Fig. 2. Serum (A) estradiol and (B) progesterone concentrations from control and DHEA-treated mice. Each column represents the mean ± S.E.M. of 10 measurements from different animals. (*) Value is significantly different from control value by analysis of variance, ∗ P < 0.05. Assay was carried out twice. (C) Ovarian prostaglandin E production of control (oil injected) and DHEA-treated mice. Each column represents the mean ± S.E.M. of 10 measurements from different animals. (*) Value is significantly different from control value by analysis of variance, ∗ P < 0.05. Assays were carried out twice.

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Glutathione (umol/gr tissue)

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Fig. 3. (A) Glutathione content of ovarian tissue from control and DHEA-treated mice. (B) Lipid peroxidation index of ovarian tissue from control and DHEA-treated mice. Each column represents the mean ± S.E.M. of 10 comparisons from different animals. (*) Value is significantly different from control value by analysis of variance, ∗ P < 0.05. Assays were carried out twice.

comitantly with total GSH diminution after 20 days of DHEA treatment (Fig. 3A), lipid peroxidation was augmented (Fig. 3B) compared with controls. 3.4. Lymphocyte infiltration and immunophenotype Considering that the immune system and its network of secretory products play an active role in ovarian function, we were interested to study possible changes in the number and immunophenotype of T lymphocytes after cystogenesis induction. Thus, 10 sections from each group were used to evaluate the relationship between CD4+ and CD8+ T cell subsets in DHEA-treated and control ovaries. Immunohistochemical analysis of control sections stained with both PE-CD4+ and FITC-CD8+ T cell markers showed that the ratio of CD4+:CD8+ T cell was approximately equivalent (Fig. 4A and B, respectively). However, following DHEA administration, the ovarian T lymphocyte immunophenotype (CD4+ and CD8+) expressions was modified. The CD4+ T cell subset diminished (Fig. 4C), while the CD8+ subset increased (Fig. 4D). We used flow cytometry also to study the number and phenotype of T lymphocytes. We found that, in control samples over 50,000 events analyzed, 42 ± 3% were lymphocytes,

68 C.G. Luchetti et al. / Journal of Reproductive Immunology 64 (2004) 59–74 Fig. 4. Representative sections of ovarian T lymphocyte immunophenotype expression quantified by fluorescent microscopy. (A) CD4+ T cell pattern from control mouse. (B) CD8+ T cell pattern from control mouse (×100). (C) CD4+ T cell pattern from mouse injected with DHEA for 20 consecutive days. (D) CD8+ T cell pattern from mouse injected with DHEA for 20 consecutive days (×100).

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while in treated samples, for the same region over 50,000 total events, 64 ± 2% were lymphocytes, indicating a significant (P < 0.001) increase in T lymphocyte infiltration. Fig. 5 illustrates a representative analysis of the 10 controls and 10 DHEA-treated samples using forward scatter (cell sizes) and side scatter (granularity) parameters. On dot plot analysis, Fig. 5A shows that in controls, CD4+ T lymphocytes represented 51 ± 3% of the

Fig. 5. Flow cytometry analysis using forward (FSC: cell size) and side scatter (SSC: cell complexity) parameters and dot plot analysis using both standard fluorescence: FL1, fluorescein (FITC) anti-mouse-CD8+ T lymphocyte; and FL2, phycoerythrin (PE) anti-mouse-CD4+ T lymphocyte. (A) A representative analysis of ovariant tissue suspension obtained from control mice. (B) A representative analysis of ovarian tissue suspension obtained from mice injected with DHEA for 20 days.

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total T cells while CD8+ T cell were was the 49 ± 4%. Cystogenesis induction with DHEA diminished the population of CD4+ T cell subset (17 ± 5%) but increased the CD8+ T cell subset (83 ± 4%) (Fig. 5B), as evaluated by Winmdi 2.8 software.

4. Discussion Part of the difficulty in understanding PCOS and interpreting the literature surrounding it, is that there is no universally accepted clinical definition. Over the years, it has evolved from “disease” to “syndrome”, the latter including a variety of potential signs and symptoms. For these reasons, it has been difficult to reproduce a similar animal model in order to compare with the human aspects. In the present report, we have used daily injection of DHEA to female prepuberal BALB/c mice to induce cystogenesis. First, we evaluated the time of apparition of cysts, which were found on day 20 of DHEA injection. This is in agreement with Anderson et al. (1997) who, using the same murine model, found that progressive DHEA treatment yielded cyst development associated with resumption of meiosis as a major initial mechanism. The ovarian morphology of our animal model showed an increased number of degenerated follicles at earliest stages and enhanced proportion of stroma and fat tissues. The number of atretic follicles were higher than those observed in normal ovaries. This appearance was similar to that described in women with PCOS (Webber et al., 2003). Our results were also coincident with the human pathology in the increase of the leukocyte infiltration (Bukulmez and Arici, 2000). On the other hand, we observed also that both serum E and P levels were augmented with induction of cystogenesis. These findings are in accordance with other authors (Lee et al., 1991; Wickenheisser et al., 2000; Fassnacht et al., 2003) who have documented increases in both cytochrome P450 17 ␣-hydroxylase and steroidogenic acute regulatory protein (StAR) activities in theca cells from women with PCOS, suggesting a global enhancement of steroidogenesis. Therefore, studies on cultures of human theca cells derived from follicles isolated from the ovaries of PCOS and normal women demonstrated that PCOS theca cells produce greater amounts of testosterone, 17 ␣-hydroxyprogesterone and progesterone than normal theca cells (Strauss, 2003). Although the mechanism for LH hypersecretion described in human ovarian polycystic pathology is not entirely clear, recent data suggest that it involves impaired negative feedback on LH secretion mediated by either high E or P levels in women with PCOS (Egleson et al., 2000). However, just how the treatment of DHEA (or the increased levels in women with PCOS) brings about such a cascade of hormonal events producing ovarian failure remain unknown. At the moment, we have focused experiments to clarify this point. This intricate network of ovarian regulators make it difficult to explain why animals with induced cystogenesis showed high levels of P but stayed in the estrus stage of the sexual cycle. As we expected, ovarian PGE production increased after DHEA treatment. Navarra et al. (1996) reported previously that granulosa cells from patients with polycystic ovaries produced a greater amount of PGE2 than that released by cells from normally ovulating women. Moreover, it was reported that increased levels of circulating PGE2 was able to regulate immune cells (Lakier, 2003; Kuroda and Yamashita, 2003; Yang et al., 2003). Thus, the enhanced ovarian PGE levels observed here may be analyzed as a consequence of the en-

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hanced lipidic metabolism reported in PCOS (Abbott et al., 2002), but the relationship between this prostanoid and the T lymphocyte response must not be overlooked. We have to take into account that PGE could contribute to the increase in recruitment of lymphocytes to ovarian cystic tissue observed in the present study, as reported in other systems (Gutzmer et al., 2004; Vancheri et al., 2004). The cystogenesis produced important damage in ovarian, as evidenced by the increase in lipid peroxidation index accompanied with significant diminution on glutathione concentration and, as far as we know, this is the first study reporting an imbalance in oxidant–antioxidant parameters in ovaries with cyst. Oxidative stress has been implicated in polycystic kidney pathology by Maser et al. (2002) who reported an increased oxidant injury concomitant with a diminution of mRNA expression of the main enzyme involved in glutathione synthesis: glutathione peroxidase. Considering the active role of DHEA on the immune system (Araghi-Niknam et al., 1997; Hernandez-Pando et al., 1998; Carr, 1998; Zhang et al., 1999; Ben et al., 1999; Du et al., 2001), we were also interested in studying possible changes in ovarian T lymphocyte expression during cystogenesis. Thus, ovarian T lymphocyte infiltration and phenotype were evaluated by both immunohistochemical and flow cytometry assays. The apparition of cysts increased ovarian T lymphocyte infiltration while diminished the populations of CD4+ and increased the CD8+ T subset. Recently, Lu et al. (2002) found that, after estrogen stimulation, there was a direct relationship between CD8+ enriched T cell population expression and high B cell-produced cytokine levels in rhesus macaque ovaries. In addition, selective changes in lymphocyte subtype were also reported in premature ovarian failure (Chernyshov et al., 2001). These findings, coupled to the fact that immune mechanisms were reported to regulate folliculogenesis (Brannstrom and Enskog, 2002), led us to suggest that in cystogenesis, the alterations in lymphocyte expression could be related with follicular atresia. Moreover, the enriched CD8+ T cell expression could be involved in the high levels of cytokines, such tumor necrosis factor, reported to be increased in the cystic pathology (Araya et al., 2002; Korhonen et al., 2002; Peral et al., 2002; Sayin et al., 2003; Deshpande et al., 2000; Gallinelli et al., 2003). At the moment, our studies are being conducted to clarify whether this selective T lymphocyte infiltration could be involved in the increase of levels of cytokines. In summary, the present data are consistent with the hypothesis that endocrine and immune responses are related in ovarian tissue.

Acknowledgements The authors thank Silvia Zorz for reviewing this manuscript. These studies were supported by Beca Ramon Carrillo-Arturo Oñativia, Ministerio de Salud (Ref. 0143) and PIP CONICET 2529.

References Abbott, D.H., Dumesic, D.A., Franks, S., 2002. Developmental origin of polycystic ovary syndrome—a hypothesis. J. Endocrinol. 174, 1–5.

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