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Articles |
Stimulates Proliferation of Rat Ovarian Theca-Interstitial Cells1
a Karol Marcinkowski University of Medical Sciences, Department of Gynecology and Obstetrics, 60-535 Poznan, Poland
b Yale University School of Medicine, Department of Obstetrics and Gynecology, New Haven, Connecticut 06520-8063
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) is a potent modulator of ovarian function, affecting steroidogenesis of both granulosa and theca-interstitial (T-I) cells. Women with polycystic ovary syndrome (PCOS) have increased levels of serum TNF-. The present study evaluated the effects of TNF- on T-I cell proliferation. Purified rat T-I cells were cultured for 48 h with or without TNF- (0.001–1 nM), insulin-like growth factor I (IGF-I; 10 nM), and/or insulin (10 nM). Proliferation was measured by [3H]thymidine incorporation assay and by counting the steroidogenically active (stained positive for 3ß-hydroxysteroid dehydrogenase; 3ß-HSD) and inactive (3ß-HSD negative) cells. TNF- stimulated thymidine incorporation in a dose-dependent fashion (up to 3.2-fold; P < 0.01). Insulin and IGF-I stimulated T-I proliferation (respectively, by up to 2.4- and 3.1-fold; P < 0.001). TNF- potentiated effects of insulin and IGF-I in a dose-dependent and additive fashion (up to 6.7-fold; P < 0.001). TNF- (1 nM) increased total cell count (by 80%, P < 0.05) and the proportion of 3ß-HSD-positive cells (by 19%, P < 0.05). Flow cytometry DNA analysis revealed that TNF- (1 nM) increased the proliferative index by up to 16% (P = 0.05). The present findings demonstrate that TNF- stimulates mitotic activity of T-I cells by increasing the proportion of actively dividing cells and preferentially increasing the number of steroidogenically active cells. The effects of TNF- appear to be independent of those induced by insulin and IGF-I. We postulate that TNF- may play a pathophysiologic role in disorders of the T-I compartment, such as PCOS.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Persuasive arguments point at insulin and insulin-like growth factors (IGF-I and IGF-II) as the key paracrine/autocrine regulators of T-I function [6, 12, 14–17]. Our recent studies have demonstrated that insulin and IGFs are potent stimulators of T-I cell proliferation in rats and humans [16,17]. Morphological and functional abnormalities of the T-I compartment in PCOS patients are likely related to insulin resistance and compensatory hyperinsulinemia, as well as to increased levels of bioavailable IGF-I.
Recent reports have demonstrated that women with PCOS have increased serum levels of tumor necrosis factor- (TNF-) [18, 19]. Furthermore, TNF- is overexpressed in models of obesity and insulin resistance in adipose tissue of rodents and human [20–22]. It is likely that TNF- contributes to development of insulin resistance and interferes with insulin signaling by induction of serine phosphorylation of insulin receptor substrate-1 and consequent inhibition of tyrosine kinase activity [23, 24].
Growing evidence indicates that TNF- is involved in regulation of normal ovarian function. TNF- immunoreactivity and mRNA have been detected in oocytes and granulosa cells of healthy and atretic antral follicles in rats and humans as well as in a subpopulation of human theca cells [25–29]. TNF- is secreted locally by granulosa-luteal cells and possibly by ovarian macrophages and lymphocytes [26, 30]. Several studies have shown that TNF- plays an important role in follicular and luteal development, ovulation, and modulation of theca and granulosa steroidogenesis. TNF- was shown to inhibit LH-stimulated androgen production by lowering LH receptor number, cAMP production, and protein kinase A activity [31–34]. TNF- also induces characteristic clustering of theca cells in culture; this action is thought to be mediated via protein kinase C pathway [35, 36]. While the physiologic significance of clustering of T-I cells remains uncertain, Zachow et al. [35] postulated that this effect may reflect an action of TNF- as an organizing factor, involved in the process of follicular development. Thus, TNF--induced clustering may play a role in the formation of the theca interna layer. Development of theca interna also requires mechanisms regulating cell proliferation and differentiation.
The present study was designed to evaluate the effects of TNF- on proliferation of T-I cells and on the proportion of cells expressing steroidogenic activity. In addition, the effects of TNF- on insulin/IGF-I-induced proliferation of T-I cells were evaluated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The following materials were purchased from Sigma Chemical Co. (St. Louis, MO): Medium 199 with Hanks' Balanced Salt Solution (HBSS) and sodium bicarbonate, Medium 199 with HBSS (10-strength), McCoy's 5a medium (modified, with sodium bicarbonate), L-glutamine, BSA, trypsin-EDTA (0.05%/0.02%), nitroblue tetrazolium grade III crystalline, estradiol-17ß, 5ß-androstan-3ß-ol-17-one, ß-nicotinamide adenine dinucleotide-oxidized form (ß-NAD+), sesame oil, paraformaldehyde, Percoll, ribonuclease, propidium iodide, ethanol, human recombinant IGF-I, and bovine insulin. Human recombinant TNF- and anti-human TNF- neutralizing antibody were obtained from R&D Systems (Minneapolis, MN). 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). The following materials were purchased from Grand Island Biological Co. (Grand Island, NY): trypan blue stain (0.4%; w:v), antibiotic-antimycotic preparation (penicillin, 10 000 IU/ml; streptomycin, 10 000 µg/ml; amphotericin B, 25 µg/ml), and Dulbecco's PBS (single-strength, pH 7.2, without MgCl2 and CaCl2). Hepes was purchased from American Bioanalytical (Natick, MA). Radiolabeled [3H]thymidine (92.0 mCi/mmol) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ; formerly Amersham Life Sciences, Arlington Heights, IL).
Animals
Immature (25 days old) female Sprague-Dawley rats were obtained from Taconic Farms (Germantown, NY) and housed with a 12L:12D photoperiod in an air-conditioned environment. Standard rat chow and water were given ad libitum. Starting on Day 28 of age, the animals were injected with estradiol-17ß (1 mg/0.3 ml sesame oil s.c.) daily for 3 days in order to stimulate ovarian development. On the morning after the last injection (Day 31 of age), the animals were anesthetized with ketamine and xylazine (i.p.) and killed 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, and with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
Isolation of T-I Cells and Cell Cultures
Ovaries were dissected, and T-I cells were isolated and purified as described previously [16]. The cells were counted using a hemocytometer, and viability was determined using trypan blue stain exclusion test. Cell viability was in the 85–95% range. The cells were cultured in McCoy's 5a medium supplemented with L-glutamine (2 mM), BSA (1 mg/ml), penicillin (10 000 IU/ml), streptomycin (10 000 µg/ml), and amphotericin B (25 µg/ml) with or without TNF- and/or IGF-I and insulin. Cultures were carried out at 37°C in an atmosphere of 5% CO2 in humidified air in 6-well plates, 24-well plates (Falcon, Becton Dickinson Labware, Lincoln Park, NJ), or 96-well plates (Corning Glass Works, Corning, NY) for up to 48 h. The final amounts of T-I cells plated were 600 x 103 cells/2 ml in 6-well plates, 350 x 103 cells/ml in 24-well plates, and 35 x 103 cells/0.25 ml in 96-well plates.
At the end of the culture period, the viability of the attached cells was in the 90–95% range. Immunohistochemical evaluation of the cells was performed before the plating and at the end of the culture period. The two cell populations had comparable staining to a mesenchymal marker, vimentin (90–95%); epithelial marker, cytokeratin (5–10%); and endothelial marker, factor VIII (< 3%). Details of the immunohistochemical methodology were presented previously [16].
Determination of DNA Synthesis
DNA synthesis of T-I cells was evaluated by radiolabeled thymidine incorporation assay whereby [methyl-3H]thymidine (4 µCi/ml) was added to cultured T-I cells during the last 24 h of culture. At the end of the culture period, the cells were harvested using a multiwell cell harvester (PHD Harvester, Model 290; Cambridge Technology, Inc., Watertown, MA). Radioactivity was measured in a liquid scintillation counter, SL 4000 (Intertechnique, Fairfield, NJ). Each treatment was carried out in at least 6 replicates.
Cell Counting and Identification of Steroidogenically Active Cells
T-I cells were cultured in 24-well plates. Each treatment was carried out in at least three replicates. At the end of the cell culture period, the cells were washed with PBS (single-strength, pH 7.2) and harvested with the aid of trypsin-EDTA solution (0.05% and 0.02%, respectively; 0.3 ml/2 cm2). Subsequently, the T-I cells were washed with PBS (single-strength, pH 7.2) and fixed in 1% paraformaldehyde for 20 min. Steroidogenically active T-I cells were identified histochemically by detection of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) activity as described previously [16, 37, 38]. Briefly, T-I cells were reconstituted in histochemical staining solution (PBS, single-strength, supplemented with 0.1% BSA, 1.5 mM ß-NAD+, 0.25 mM nitroblue tetrazolium, and 0.2 mM 5ß-androstan-3ß-ol-17-one) and incubated overnight at 37°C in the dark. Under these conditions, 5ß-androstan-3ß-ol-17-one served as a substrate to 3ß-HSD; blue staining of the cells was the effect of the subsequent redox reaction with nitroblue tetrazolium. The number of stained cells (steroidogenically active) and nonstained cells (steroidogenically inactive) was determined using a hemocytometer by counting 10 squares from each sample. Prior to cell counting, the samples were randomized, and the observer counting the cells was blinded to the treatment.
Flow Cytometry Analysis of Cell Cycle
Assessment of the cell cycle distribution was performed by flow cytometry of propidium iodide (PI)-stained T-I cells according to a modified method described by others [39]. Initially, T-I cells were cultured in 6-well plates in serum-free McCoy's 5a medium for 24 h. Subsequently, the medium was changed and T-I cells were incubated with or without TNF- (1 nM) for 8 or 24 h. At the end of the culture periods, the cells were dispersed with the aid of trypsin-EDTA solution and washed. Samples containing approximately 106 cells were resuspended in 2 ml of ice-cold PBS and fixed by three stepwise additions of 2 ml each of 95% ice-cold ethanol. The fixed T-I cells were stored at 4°C awaiting flow cytometry analysis. On the day of analysis, the T-I cells were spun down and resuspended in ribonuclease solution (1 mg/ml, in PBS, pH 7.0) for 30 min at 37°C and subsequently treated with PI (0.05 mg/ml) for 1 h. Prior to the analysis, the samples were filtered through 35-µm nylon mesh (TETKO Inc., Elmsford, NY). Flow cytometric analysis was performed with a FACS Vantage flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). The cells were excited with an argon laser operating at 488 nm. PI fluorescence was collected in a linear mode through a 630/22-nm band pass filter. At least 15 000 cells were analyzed for each sample. Cell cycle analysis was performed using the Modfit 5.2 multitrapezoid model (Verity Software House, Topsham, ME). The results were expressed as proliferative index (the sum of percentages of cells in S and G2/M phases of the cell cycle obtained from DNA histograms).
Statistical Analysis
Results are presented as the mean ± SEM, unless stated otherwise. Comparisons between the means were performed using ANOVA followed by post hoc comparisons of individual means using Bonferroni correction. In the absence of normal distribution, the data were analyzed by Kruskal-Wallis nonparametric test and presented in the form of box plots.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on DNA Synthesis
TNF- stimulated DNA synthesis in a dose-dependent fashion (Fig. 1A). A significant increase in thymidine incorporation was observed at TNF- concentrations 
0.01 nM, with the maximal stimulatory effect (3.2-fold of control level) observed at the highest dose (1 nM; P < 0.001).
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In order to determine whether TNF- modulates effects of insulin and IGF-I, T-I cells were cultured for 48 h in the presence of insulin (10 nM) or IGF-I (10 nM) either alone or in combination with TNF- (0.1–1 nM) (Fig. 1B). These doses of insulin and IGF-I have been previously shown to produce near-maximal stimulatory effects on T-I cell proliferation [16]. In the present study, insulin alone and IGF-I alone significantly stimulated thymidine incorporation to 2.4- and 3.1-fold, respectively, of control levels (P < 0.01). TNF- potentiated these proliferative effects of insulin and IGF-I in a dose-dependent and additive fashion. Combining TNF- (1 nM) with insulin or IGF-I, respectively, increased DNA synthesis to 5.5-fold and 6.7-fold of the control level (P < 0.001). DNA synthesis in the presence of TNF- plus insulin and TNF- plus IGF-I was significantly greater than in the presence of each of these peptides alone (P < 0.01).
Determination of the T-I Cell Number and the Presence of 3ß-HSD Activity
The effect of TNF- on T-I cell proliferation was confirmed by direct counting of the cells (Fig. 2A). At the end of 48-h culture, TNF- (1 nM) had increased the total cell number (sum of 3ß-HSD-positive and -negative cells) by 80% above the cell number in the control cultures (P < 0.05). This effect was associated with a preferential increase in the proportion of steroidogenically active cells. As presented in Figure 2B, TNF- (1 nM) increased the proportion of steroidogenically active cells (by 19%; P < 0.05).
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Effects of TNF- on T-I Cell Cycle Progression
Flow cytometry cell cycle analysis of PI-stained T-I cells was performed. The proportion of mitotically active T-I cells (proliferative index = percentage of cells in S and G2/M phases) was determined in cultures with or without TNF- (1 nM). DNA analysis showed that treatment with TNF- (P = 0.05) increased the proliferative index of T-I cells at 8 and 24 h of incubation as shown in Figure 3.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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stimulates proliferation of T-I cells in a dose-dependent fashion; 2) TNF- augments proliferative actions of insulin and IGF-I, and this effect is additive and dose-dependent; 3) TNF- predominantly increases the population of steroidogenically active cells; and 4) the proliferative effects of TNF- are due, at least in part, to an increase in the proportion of mitotically active T-I cells.
In this study, TNF- stimulated T-I cell proliferation over a broad range of concentrations (0.01–1 nM). These concentrations are comparable to those found in human follicular fluid (0.02–0.095 nM) and serum (0.03–0.06 nM) [18, 40]. The Kd of receptor sites for TNF- localized on granulosa cells is also in a comparable range (approximately 0.2 nM) [41]. In addition, other studies have demonstrated that similar levels of TNF- modulated T-I steroidogenesis and morphologic appearance of the cultures [33, 35, 36].
TNF- may act by stimulating the G0/G1 transition and T-I cell cycle progression. This concept is supported by flow cytometry analysis of DNA. Even a modest increase in the proliferative index may cumulatively lead to a significant increase of the cell number. Thus, TNF- may either act directly as a progression factor or induce the production of other growth factors responsible for activation of nuclear transcription factors.
In this study, direct counting of T-I cells confirmed the findings of thymidine incorporation experiments; furthermore, quantification of steroidogenically active and inactive cells showed that TNF- preferentially stimulates proliferation of cells possessing 3ß-HSD activity. This observation is surprising in view of a report describing inhibitory actions of TNF- on several steroidogenic enzymes, including 3ß-HSD [31]. Furthermore, TNF- inhibits LH-dependent androgen and progesterone production [31, 34]. This discrepancy may be only an apparent one, since the overall steroidogenic output of the cell culture is a function of the number of steroidogenically active cells and the level of activity of individual cells. It is therefore possible that the higher number of cells with low activity may result in a lower steroidogenesis than does a lower number of highly active cells.
To date, few investigators have evaluated effects of TNF- on T-I cell proliferation. Adashi and his associates reported no effects of TNF- (0.57 nM) on the cell number and DNA content of T-I cells and ovarian dispersates cultured in serum-free medium [32, 42]. The discrepancy between this finding and our study may be due to differences in experimental conditions; for example, the results may be affected by contamination of ovarian dispersate cultures with other cell types and their products, including granulosa cells, ovarian macrophages, and lymphocytes. In another experimental model using purified T-I cells, Zachow and Terranova [36] did not detect any significant effect of TNF- (0.057 and 0.57 nM) on the T-I cell number after a 6-day culture. This report contrasts with our finding of almost doubling of the T-I cell number after a 48-h culture with a comparable concentration of TNF- (1 nM). These discordant findings may be due to differences in the selection of animal models. Zachow and Terranova used hypophysectomized immature rats, whereas in our studies, intact immature rats received estradiol for 3 days. Ovaries from hypophysectomized rats are characterized by a marked increase in follicular atresia [43], which may affect the proliferative potential of T-I cells. On the other hand, in vitro administration of estrogen to hypophysectomized rats has been shown to potentiate gonadotropin-induced follicular development [44]. In the present animal model, estradiol administration to intact rats enhanced T-I proliferation in response to insulin and IGF-I (results not shown). It is difficult to speculate whether this model or the hypophysectomized rat model is more physiological.
TNF- has a diverse spectrum of biologic activities, including stimulation of cell proliferation, differentiation, and induction of apoptotic death. The ability of TNF- to exert a wide variety of effects is likely due to actions via multiple signaling pathways involving two distinct receptors: TNF--R1 (55 kDa), predominantly responsible for transduction of the death signal, and TNF--R2 (75 kDa), chiefly implicated in cell proliferation [45]. It appears that in the ovary, TNF- can trigger either proliferation or apoptosis depending on the cell type and the stage of follicular development; these effects may also be species specific. Thus, for example, TNF- stimulates proliferation in human granulosa-luteal cells, while it induces apoptosis in granulosa cells of early antral follicles from rats [46–48]. To the best of our knowledge, there are no studies identifying the TNF- receptor and its specific subtypes in T-I cells. The present finding of TNF--induced proliferation in T-I cultures suggests that this effect is likely mediated via TNF--R2 receptors.
TNF- acts via multiple signal transduction pathways; for example, inhibition of LH-dependent steroidogenesis of T-I cells is mediated by the cAMP/protein kinase A pathway, whereas T-I clustering appears to be due to activation of epidermal growth factor tyrosine kinase and the subsequent increase in protein kinase C activity [36]. The role of these pathways in the induction of T-I cell proliferation by TNF- is not known. The additive nature of TNF- and insulin/IGF-I in the stimulation of T-I proliferation indicates that these agents do not share a common signal transduction pathway. Notably, the concentrations of insulin and IGF-I used in this study have been previously shown to produce near-maximal effects on DNA synthesis; furthermore, the effects of insulin and IGF-I observed in this and in the previous studies were comparable [6, 16].
TNF--induced proliferation of T-I cells may have important clinical implications. It is tempting to speculate that under pathological conditions TNF- may be responsible for excessive proliferation of the T-I compartment. Hyperplasia of the theca and stroma compartment and the overall increase in ovarian size are characteristic features of common endocrinopathies: PCOS and hyperthecosis. Indeed, serum levels of TNF- are elevated in women with PCOS when compared with normal controls [18, 19]. Local intraovarian production of TNF- by resident macrophages and leukocytes may be also increased. This hypothesis is supported by the observation of a high number of white blood cells in PCOS ovaries [49]. Moreover, TNF- production by murine macrophages and human monocytes is modulated by IGF-I, a growth factor implicated in the pathogenesis of PCOS. IGF-I has been shown to increase mRNA expression and secretion of TNF- [50].
An additional argument for the role of TNF- in the pathogenesis of PCOS is related to insulin resistance and compensatory hyperinsulinemia characteristic of this condition. TNF- is overexpressed in models of obesity and insulin resistance, interferes with insulin signaling, and induces insulin resistance [20–23, 51].
In summary, our findings indicate that TNF- has a mitogenic effect on T-I cells and preferentially increases the proportion of steroidogenically active cells. TNF- augments proliferative effects of other growth factors, such as insulin and IGF-I, and may play an important role in the regulation of T-I cell function. Under pathologic conditions, excessive stimulation by TNF- may contribute to development of hyperplastic disorders of the theca-stromal compartment, such as PCOS and hyperthecosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Antoni J. Duleba, Yale University School of Medicine, Department of Obstetrics and Gynecology, 333 Cedar Street, New Haven, CT 06520. FAX: 203 785 7134; antoni.duleba{at}yale.edu
Accepted: May 17, 1999.
Received: February 22, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) in polycystic ovary syndrome (PCOS). In: 44th Annual Meeting of Soc Gynecol Investig; 1997; San Diego, CA. on luteinizing hormone (LH)/-insulin-like growth factor-I (IGF-I)-regulated androstenedione biosynthesis and IGF-I directed LH receptor number in cultured ovarian theca-interstitial cells. Endocrine 1994; 2:1145–1150. production. Endocrinology 1996; 137:4611–4618.[Abstract]This article has been cited by other articles:
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  E. H. Ruder, T. J. Hartman, J. Blumberg, and M. B. Goldman Oxidative stress and antioxidants: exposure and impact on female fertility Hum. Reprod. Update, June 4, 2008; (2008) dmn011v1. [Abstract] [Full Text] [PDF]
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 D. J. Skarzynski, I. Woclawek-Potocka, A. Korzekwa, M. M. Bah, K. Piotrowska, B. Barszczewska, and K. Okuda Infusion of Exogenous Tumor Necrosis Factor Dose Dependently Alters the Length of the Luteal Phase in Cattle: Differential Responses to Treatment with Indomethacin and L-NAME, a Nitric Oxide Synthase Inhibitor Biol Reprod, April 1, 2007; 76(4): 619 - 627. [Abstract] [Full Text] [PDF]
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 F. Gonzalez, N. S. Rote, J. Minium, and J. P. Kirwan Increased Activation of Nuclear Factor {kappa}B Triggers Inflammation and Insulin Resistance in Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., April 1, 2006; 91(4): 1508 - 1512. [Abstract] [Full Text] [PDF]
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 V. Sander, C. G. Luchetti, M. E. Solano, E. Elia, G. Di Girolamo, C. Gonzalez, and A. B. Motta Role of the N, N'-dimethylbiguanide metformin in the treatment of female prepuberal BALB/c mice hyperandrogenized with dehydroepiandrosterone. Reproduction, March 1, 2006; 131(3): 591 - 602. [Abstract] [Full Text] [PDF]
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F. Gonzalez, N. S. Rote, J. Minium, and J. P. Kirwan Reactive Oxygen Species-Induced Oxidative Stress in the Development of Insulin Resistance and Hyperandrogenism in Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., January 1, 2006; 91(1): 336 - 340. [Abstract] [Full Text] [PDF]
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 A.J. Duleba, N. Foyouzi, M. Karaca, T. Pehlivan, J. Kwintkiewicz, and H.R. Behrman Proliferation of ovarian theca-interstitial cells is modulated by antioxidants and oxidative stress Hum. Reprod., July 1, 2004; 19(7): 1519 - 1524. [Abstract] [Full Text] [PDF]
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D. J. Skarzynski, M. M. Bah, K. M. Deptula, I. Woclawek-Potocka, A. Korzekwa, M. Shibaya, W. Pilawski, and K. Okuda Roles of Tumor Necrosis Factor-{alpha} of the Estrous Cycle in Cattle: An In Vivo Study Biol Reprod, December 1, 2003; 69(6): 1907 - 1913. [Abstract] [Full Text] [PDF]
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N. Ledee-Bataille, G. Lapree-Delage, J.L. Taupin, S. Dubanchet, J. Taieb, J.F. Moreau, and G. Chaouat Follicular fluid concentration of leukaemia inhibitory factor is decreased among women with polycystic ovarian syndrome during assisted reproduction cycles Hum. Reprod., October 1, 2001; 16(10): 2073 - 2078. [Abstract] [Full Text] [PDF]
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H. F. Escobar-Morreale, R. M. Calvo, J. Sancho, and J. L. San Millan TNF-{alpha} and Hyperandrogenism: A Clinical, Biochemical, and Molecular Genetic Study J. Clin. Endocrinol. Metab., August 1, 2001; 86(8): 3761 - 3767. [Abstract] [Full Text] [PDF]
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