Department of Obstetrics and Gynecology, University of South Dakota School of Medicine, 1400 West 22nd Street, Sioux Falls, SD 571051570, USA
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Abstract |
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Key words: granulosa cells/insulin/leptin/progesterone
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Introduction |
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Recent evidence has revealed a possible role for leptin as a direct regulator of ovarian function. Leptin inhibited insulin-induced steroidogenesis by bovine granulosa (Spicer and Francisco, 1997) and thecal (Spicer and Francisco, 1998
) cells, and suppressed insulin-like growth factor (IGF)-1 action in mediating follicle stimulating hormone (FSH) stimulation of rat granulosa cell steroid production (Zachow and Magoffin, 1997
). Leptin and its receptor are expressed in the human ovary (Cioffi et al., 1997
; Karlsson et al., 1997
), and a high dose of leptin suppressed LH-stimulated oestradiol production in human granulosa cells (Karlsson et al., 1997
). The objective of the present study was to determine whether leptin, at physiological doses, alters gonadotrophin- and insulin-stimulated progesterone production by luteinized human granulosa cells (GC) in vitro.
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Materials and methods |
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Follicular aspirates from each patient were pooled, washed in Ham's F-10 medium supplemented with 0.1% bovine serum albumin (BSA), and centrifuged over 40% Percoll (Sigma, St Louis, MO, USA) to remove red blood cells. GC were washed again with Ham's F-10/BSA, resuspended in bicarbonate-buffered Dulbecco's modified Eagle medium (DMEM)/Ham's F-12 (1:1 v/v), and plated onto 96-well plastic culture plates (CoStar, Cambridge, MA, USA) coated with bovine fibronectin (Sigma) at a density of 2x104 cells/0.2 ml. The culture medium (DMEM/F-12) was supplemented with penicillin/streptomycin (50 000 units/50 mg/l; Sigma), low density lipoprotein (25 µg/ml; Sigma), transferrin (5 mg/l; Sigma), sodium selenite (0.25 nmol/l; Sigma), and bovine insulin (2 µg/ml; Sigma). For experiment 2 (see below), human insulin (030 µg/ml) was substituted for the bovine insulin. Cells were cultured in a humidified atmosphere of 5% CO2:95° air at 37°C.
Experiment 1
As a preliminary experiment to determine whether a high dose of leptin altered progesterone production, GC (n = four patients) cultures were treated in duplicate wells with human recombinant leptin (0 or 100 ng/ml; Linco Research, St Louis, MO, USA) ± HCG (100 ng/ml; CR-127, NHPP-NIDDK, Baltimore, MD, USA) for 24 or 48 h. To further characterize the time- and dose-dependency of leptin action, GC (n = 6 patients) were cultured for up to 48 hours with leptin (0100 ng/ml) ± HCG (100 ng/ml). Progesterone concentrations in media were measured at 2, 4, 24, and 48 h.
Experiment 2
GC (n = 6 patients) were treated in triplicate wells with leptin (0 or 50 ng/ml) and human insulin (030 µg/ml; Sigma) ± HCG (100 ng/ml). Progesterone concentrations in media were measured after 1, 2, 4, and 6 days of culture.
Progesterone was assayed using Diagnostic Products Corporation (DPC, Los Angeles, CA, USA) Coat-a-Count radioimmunoassay kits. Media samples were diluted (1:100 or 1:200) in zero standard and measured in duplicate by direct assay following the manufacturer's protocol. Parallelism was demonstrated across multiple dilutions. Low, medium, and high range controls were run at the end of each standard curve and again at the end of each assay. Intra- and inter-assay coefficients of variation were 4.9 ± 1.1 and 7.7% respectively.
Data were log-transformed to achieve homogeneity of variance prior to analysis of variance (ANOVA). For experiment 1, one-way ANOVA with repeated measures blocked by patient was performed on data sets from each time point (i.e. 2, 4, 24, 48 h). For experiment 2, two-way ANOVA was performed on data sets from each time point (i.e. day 1, 2, 4, 6) with treatments (±leptin, ±HCG) as factor A and insulin concentrations as factor B. These analyses were followed by one-way ANOVA with repeated measures blocked by patient across treatments (±leptin, ±HCG) for each concentration of insulin, and across insulin concentrations within each treatment. In all cases, mean comparisons were made using Scheffé's f-test. Significance was assumed at P < 0.05.
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Results |
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Discussion |
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Inhibition of HCG-stimulated progesterone production by luteinized human GC is consistent with the results of previous studies on rat (Zachow and Magoffin, 1997), bovine (Spicer and Francisco, 1997
), and human (Karlsson et al., 1997
) GC demonstrating leptin suppression of gonadotrophin-stimulated oestradiol production. That physiological doses of leptin (1050 ng/ml) suppressed progesterone production by as early as 4 h in culture indicates that leptin can act in an acute manner. Moreover, GC appear to be very sensitive to leptin action as indicated by suppression of steroidogenesis by very low doses of leptin (as little as 0.1 ng/ml).
Our initial experiment showing leptin inhibition of HCG-stimulated progesterone production was conducted with bovine insulin present in the culture medium. The second series of experiments was conducted to investigate the effect of leptin on the insulin responsiveness of human GC steroidogenesis. It was reported that leptin impaired the synergistic effect of insulin-like growth factor (IGF-1) in potentiating FSH stimulation of rat GC oestradiol synthesis, but did not alter the stimulatory effect of FSH alone (Zachow and Magoffin, 1997). Similarly, it was reported that leptin inhibited FSH-stimulated steroid production by bovine GC in the presence of insulin, but had minimal or no effect in the absence of insulin (Spicer and Francisco, 1997
). Our results confirm these animal studies in that leptin only suppressed HCG-stimulated progesterone production in the presence of insulin.
The degree of leptin inhibition varied with different concentrations of insulin and duration of treatment. However, there was not a clear dose- and time-dependent relationship. For example, the most pronounced effect of leptin was consistently at a concentration of 3 µg/ml insulin relative to higher or lower insulin concentrations. Moreover, leptin in the absence of insulin or at very low concentrations tended to enhance non-gonadotrophin-stimulated progesterone production at certain time points. Therefore the cellular signalling interactions among gonadotrophins, insulin, and leptin may be quite complex. Furthermore, we do not know what effect, if any, intrinsic IGF may have had in our culture system.
Insulin and IGF are known to enhance stimulable cyclic(c) AMP formation and enhance cAMP action within GC (Adashi, 1993). The exact mechanism by which insulin/IGF signalling pathways communicate with the adenyl cyclase pathway is not completely understood. Insulin receptor substrate-1 (IRS-1) is a ubiquitous protein in insulin/IGF-responsive cells that can act as a link between the insulin receptor and other signalling pathways (White, 1997
). Thus leptin-induced suppression of insulin action in GC might involve IRS-1. Indeed, recent evidence has demonstrated that both TNF
(Hotamisligil et al., 1996
) and leptin (Bjorbaek et al., 1997
) can modulate IRS-1 activity in adipocytes. The fact that leptin did not alter insulin-supported progesterone production in the absence of gonadotrophin strongly implicates impairment of cAMP formation as the ultimate consequence of leptin action.
Leptin has been proposed to play a role in the aetiology of polycystic ovary syndrome (PCOS) (Brzechffa et al., 1996; Micic et al., 1997
), although this possibility remains quite controversial (Gennarelli et al., 1998
). Considering the importance of insulin resistance and hyperinsulinaemia in the aetiology of PCO (Homburg et al., 1996
), antagonistic interaction between leptin and insulin at the ovarian level has intriguing implications. Although leptin may not be an obligatory factor in PCO (Gennarelli et al., 1998
), it remains to be elucidated how leptininsulin interactions in the ovary are manifested clinically.
Although leptin is primarily secreted by adipocytes, leptin mRNA expression has been reported to be present in granulosa and cumulus cells of pre-ovulatory human follicles (Cioffi et al., 1997). Moreover, in immunofluorescence studies, leptin exhibited a localized distribution in cumulus cells associated with regions of intense leptin immunofluorescence in the oocyte (Antczak and Van Blerkom, 1997
). These authors proposed that leptin from cumulus cells may be transferred into oocytes and play a role in embryonic development. Thus leptin may have multiple, complex roles in ovarian function.
In summary, leptin at physiological levels suppresses gonadotrophin-stimulated progesterone production by luteinized human GC apparently through antagonism of insulin (and/or IGF) action. The cellular mechanism of this antagonism remains to be elucidated. The results suggest that leptin may be a physiological regulator of follicular and/or luteal function in humans.
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Acknowledgments |
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Notes |
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References |
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Submitted on October 28, 1998; accepted on February 15, 1999.