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Regular Article |
a Department of Obstetrics and Gynaecology and Department of Physiology, St. George's Hospital Medical School, Tooting, London SW17 0RE, United Kingdom
b Department of Surgery, Bristol Royal Infirmary, Bristol University, Bristol BS2 8HW, United Kingdom
c Department of Obstetrics and Gynaecology, University of Malta Medical School, Msida, Malta, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IR3 or excess IGFBP-3 to remove the effects of endogenous IGF action. Steroid accumulation in the medium was assessed. IGFBP-4 continued to exert potent inhibitory effects when the action of endogenous IGF was removed from the system, demonstrating that its actions are independent of IGF binding. There was no effect on cell metabolism, and the effects on steroidogenesis were reversible after IGFBP-4 removal from the culture medium. No similar effects were seen with IGFBP-2. These reasults are the first evidence of IGF-independent IGFBP-4 actions and the first evidence of IGF-independent actions of any IGFBPs in the ovary.
granulosa cells, growth factors, insulin-like growth factor receptor, ovary, steroid hormones
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In an earlier study, we demonstrated partial inhibition of FSH-stimulated steroidogenesis by incubation of granulosa cells with IGFBP-1 or -3, which we suggested was due to the sequestration of IGF-II in these cultures [8]. Recently, we reported an inhibitory effect of IGFBP-4 on both granulosa and theca steroidogenesis that was more potent than might be expected by sequestration of IGF alone [9] and was more potent than that previously seen with IGFBP-3 or -1. We hypothesized that the effects of IGFBP-4 in these experiments are independent of its ability to bind IGF. The series of experiments reported here were designed to test this hypothesis
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries were obtained from women undergoing oophorectomy for nonovarian gynecological disease. All patients were cycling regularly except one who reported polymenorrhea and one with oligomenorrhea. Cycle stage was random, and none of the patients had received medication for stimulation or suppression of ovarian function for at least 3 mo prior to surgery. Patient details are given in Table 1. Approval for this study was granted by the local ethics committes and by the Ethics Committee of the Faculty of Medicine and Surgery, Medical School, Malta. Informed consent was obtained from each patient prior to surgery. Morphological category was assigned according to previously published criteria [10]. Most of the ovaries collected were ovulatory and polycystic.
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Cell Cultures
Follicles were microscopically dissected intact from the ovaries, the diameter was measured, and follicular fluid was aspirated. Granulosa cells were harvested and cultured as previously described [11]. Experiments were carried out on pooled cells from a single patient or on cells from individual follicles where stated. In one experiment, cells were pooled from two patients. Details of the size of follicles from which cells were pooled is shown in Table 1. Approximately 5 x 104 viable cells/well were incubated in a 200-µl volume of serum-free Medium 199 (Gibco BRL, Paisley, U.K.) with the addition of antibiotics (penicillin and streptomycin; Gibco BRL) and 200 mM L-glutamine. Incubations were carried out in the presence of 10-7 M testosterone (Sigma Chemical Co., Poole, U.K.) as an aromatase substrate. One experiment was carried out using cells collected at the time of oocyte aspiration from patients undergoing ovarian stimulation for in vitro fertilization. Following removal of the oocyte, aspirates were pooled and granulosa cells were separated from blood cells and other debris using the method of Abeyasekara et al. [12].
Experimental Protocol
Experiments were performed on cells from six patients, two of whom had normal ovaries and four of whom had ovulatory polycystic ovaries. Cells were incubated in medium alone or in medium containing 5 ng/ml highly purified human pituitary LH or FSH (Endocrine Services, Bidford-on-Avon, U.K.) with or without IGFBP-4 (Austral Biologicals, San Ramon, CA) at 5 or 50 ng/ml, doses that previously caused maximum inhibition of steroid production [9]. All cultures were performed with the addition of 5 x 10-7 M testosterone as an aromatase substrate.
Although the majority of these experiments were conducted with cells from small follicles in which the levels of endogenous IGF would be expected to be very low or absent, to negate the contribution of any endogenous IGF to the steroid production, experiments were carried out in the presence of the specific IGF-I receptor monoclonal antibody IR-3 (Oncogene Science, Cambridge, U.K.) [13], which is purified on a protein-G affinity column. The concentration of antibody used (100 ng/ml) had previously been determined to completely inhibit the stimulatory effects of addition of 10 ng/ml IGF-I in granulosa cells [2]. A control to demonstrate this inhibition was added to each experiment.
To further demonstrate IGF-independent effects, a second series of experiments was performed in which IGFBP-4 was added in the presence of 50 ng/ml IGFBP-3, a concentration calculated to bind any endogenous IGF-II, previously found to be <10 ng/ml [4]. In addition to IGFBP-4, IGFBP-2 is also selectively cleaved in follicular fluid from healthy follicles, and in a further experiment the effects of IGFBP-2, -3, and -4 were compared in the same pool of cells. To investigate the degree of fragmentation of the IGFBPs under these experimental conditions, medium was subsequently subjected to immunoblot for IGFBP-3 and -4, and the percentage of fragmentation was calculated after densitometric analysis of bands representing intact and fragmented IGFBP.
All treatments were added simultaneously. Cultures were incubated for 48 h, the medium was collected, and estradiol (E2) and progesterone accumulations were measured with an in-house RIA, as previously described [14, 15]. This nonextraction method was specifically designed for use with culture medium, using antibodies supplied by Guildhay (Guildford, Surrey, U.K.).
We also performed two experiments to determine the permanency of the IGFBP-4 inhibitory effects and to see whether IGFBP-4 also affected cell viability or metabolism. To investigate the reversibility of IGFBP-4 effects, one set of cells was incubated with FSH with and without IGFBP-4 for 24 h, after which the medium was replaced by medium containing LH alone for a further 24 h. To test for possible effects of IGFBP-4 on cell viability, medium was removed and cells were exposed to a colorimetric assay in which the cells are incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma). The assay gives a qualitative assessment of mitochondrial dehydrogenase enzyme activity, which converts MTT to dark blue formazon. The resultant color change measured by optical density is proportional to the metabolic activity and number of cells [16].
Each test substance was added to three to six wells. Results are expressed as the mean and standard error. Differences between treatments were assessed with a Mann-Whitney U-test, with significance assumed at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Significant inhibition of basal steroid production by IGFBP-4 was seen in three of five experiments performed in various pools of cells from three different patients (Fig. 1, a–c, first two bars). The degree of inhibition of both progesterone and E2 was variable. In the same experiments, gonadotropin stimulated steroid production, which was inhibited by IGFBP-4 in all experiments. Figure 1a shows progesterone results from granulosa cells from a single follicle 12 mm in diameter. Similar results were obtained for E2 in the same wells. Results for E2 from granulosa cells pooled from small follicles from the same ovary are shown in Figure 1b and those for cells from small follicles from a second ovary are shown in Figure 1c. The results for progesterone in these experiments were similar to those for E2.
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Addition of IR-3 in these experiments had a slight agonistic or no effect on basal and gonadotropin-stimulated E2 and progesterone production, demonstrating that endogenous IGF was either absent or at a very low level. Likewise, addition of IR-3 in the presence of FSH had little effect on the level of steroid, indicating that neither FSH nor LH stimulated IGF-II production in these experiments and that endogenous IGF did not contribute significantly to the steroid response. The effectiveness of the antibody in this system was demonstrated by the fact that addition of 100 ng/ml of IR-3 completely inhibited steroid production stimulated by addition of IGF-I.
Incubation of cells in the presence of IR-3 did not affect the inhibitory action of IGFBP-4 on FSH-stimulated steroidogenesis (Fig. 1, a–c, last bars). Similar results were obtained for E2 and progesterone in cells pooled from small follicles from a third pair of ovaries (data not shown).
The results of addition of IGFBP-4 in the presence of IGFBP-3 on FSH-stimulated E2 and progesterone production are shown in Figure 2, a and b. IGFBP-4 inhibited the stimulatory effects of FSH in each case. Coincubation with IGFBP-3 had no significant effect on FSH-stimulated steroid production in cells from these small follicles, again suggesting that the levels of endogenous IGF were low. IGFBP-4 continued to cause inhibition of E2 and progesterone even in the presence of IGFBP-3, which had previously been shown to negate the effects of IGF-I [8]. Similar results were obtained in luteinized cells collected as a by-product of oocyte aspiration in patients undergoing egg collection for in vitro fertilization (Fig. 2c).
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Recovery of Steroidogenic Capacity FollowingIGFBP-4 Treatment
In this experiment, IGFBP-4 inhibited LH-stimulated progesterone production during the first 24 h of incubation (Fig. 3). After the medium was removed and the cells were restimulated with LH, there was complete recovery of progesterone production to levels observed in cells that had not been exposed to IGFBP-4, and there was partial recovery of E2 production.
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Comparative Effects of IGFBP-2, -3, and -4
Comparison of the effects of IGFBP-2, -3, and -4 revealed that only IGFBP-4 significantly inhibited granulosa cell E2 production (Fig. 4). In cells from this pool of follicles, IGFBP-2 had no effect and IGFBP-3 caused a slight but not significant inhibition of steroidogenesis. Western immunoblotting of media containing exogenous IGFBP-2 and -3 revealed that the vast majority of the binding protein remained in the intact form (data not shown). This finding was particularly apparent for IGFBP-2, where <1% was fragmented.
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MTT Results
There was no significant difference between any of the treatments in the optical density of medium in the wells in the MTT assay.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IR-3 known to block the effects of endogenous IGF. The IR-3 antibody is highly specific for and binds with high affinity to the IGF-I receptor. Because IGF-II is known to exert its effects via this receptor [2] and IGF-I is not made by these cells [17], all effects of IGF should be negated in these experiments. The lack of effects of IR-3 and IGFBP-3 in our experiments however demonstrates that the levels of endogenous IGF were very low in these cultures. This finding suggests that IGFBP-4 is acting on these cells in a manner independent of its ability to sequester IGF. This is the first demonstration of IGF-independent effects of IGFBP-4 and the first demonstration that IGFBPs may have IGF-independent effects in the ovary. There was significant inhibition of basal steroid production in approximately half of our experiments, whereas IGFBP-4 consistently inhibited gonadotropin-stimulated steroid production. There were no consistent differences in the composition of follicle pools that could account for the inconsistency in basal inhibition.
IGF-independent effects of IGFBPs have been demonstrated in a variety of cells types, but to date these effects have been shown only for those IGFBPs known to interact with cell surface or extracellular matrix binding sites. For example, IGFBP-1 stimulates the migration of smooth muscle cells by a mechanism involving binding to the integrin receptor [18]. Nuclear localization of IGFBP-3 and -5 has been recently reported, presenting an intriguing possibility for the mechanism of action of IGFBPs in some cell types [19, 20]. At present, we have no data to suggest a mechanism by which IGFBP-4 may be exerting its effects.
The majority of women undergoing total abdominal hysterectomy and bilateral salpingoophorectmy have ovulatory polycystic ovaries (personal observation). Although cells from these ovaries are poor responders to gonadotropin [10] and are therefore not ideal for experimental purposes, the supply of tissue is not such that all studies can be performed with normal ovaries. We considered the use of polycystic tissue for these studies valid because all of the pools of cells responded to gonadotropin and because we were not investigating the differential effects of IGFBP-4 in normal versuis polycystic ovaries, only on unluteinized granulosa cells in general.
The inhibitory effects of IGFBP-4 were confined to this binding protein alone; there was no or limited inhibition of E2 and progesterone production by IGFBP-3 or IGFBP-2 in these cells. IGF-I is not produced by human granulosa cells, and according to previous work the IGF-II mRNA is not detectable until the follicle reaches 11 mm in size, i.e., following selection [1]. There are data, however, to suggest that IGF-II production may be stimulated in response to FSH and other factors in these cells [21, 22], which may explain the limited effect of IGFBP-3 on these cells. In our experiments, IR-3 had little or no effect even in the presence of gonadotropin, indicating that the levels of endogenous IGF were low throughout. Proteolysis of IGFBP-2 in follicular fluid from healthy follicles has been reported consistently and appears to be a prerequisite for folliculogenesis; however, our data indicate that this proteolysis does not appear to be due to the antigonadotropic effects of IGFBP-2. We have been unable to detect IGFBP-3 in granulosa cell-conditioned medium, and although IGFBP-2 was present it was always entirely in the intact form [7]. In the experiments reported here, the vast majority of the binding protein remained in the intact form at the end of the culture period, demonstrating that the differences in inhibitory activity of these IGFBPs cannot be due to differences in fragmentation in the medium. Further work is required to elucidate the role of IGFBP-2 in the ovary.
The degree to which IGFBP-4 was antigonadotropic in these cells emphasizes the physiological reason for its removal from healthy follicles. There was complete recovery of progesterone secretion and partial recovery of E2 secretion following removal of IGFBP-4, showing that the effects of the binding protein were reversible. IGFBP-4 does not cause cell death or toxicity, as further supported by the lack of an effect on metabolism in these cells as indicated by the results of the MMT assay. Removal of IGFBP-4 in the ovary is achieved both by suppression of mRNA production and by proteolytic cleavage. Although we and others have demonstrated IGFBP-4 proteolytic activity in follicular fluid of all sizes of healthy follicles, we have only seen activity in one sample of granulosa cell-conditioned medium, which was from a 25-mm preovulatory follicle [23] Proteolysis of IGFBP-4 in granulosa cells was induced by FSH in rat granulosa cells and by FSH and IGF-I in luteinized human granulosa cells [24, 25], but addition of IGFs in the present study had no effect on protease production by granulosa cells from small follicles.
We are currently investigating the possible presence of an inhibitor of this activity. Others have suggested that in fibroblasts at least the specific IGFBP-4 protease is pregnancy-associated plasma protein A (PAPP-A) [26]. The presence of PAPP-A has been demonstrated in follicular fluid, and levels were higher in estrogen-dominant follicles [27]. This protein and its possible role as an IGFBP-4 protease will no doubt be the focus of considerable interest in the near future.
There have been several reports of high levels of IGFBP-4 in follicular fluid from polycystic ovaries. Falling levels of IGFBP-4 were found in a spontaneous preovulatory follicle from a woman with polycystic ovaries [28]. Given the potency of the inhibitory effects of IGFBP-4 on follicular cells, it is possible to envision a role for IGFBP-4 in the apparent inability of these cells to respond to FSH in vivo. Removal of the cells from the follicular endocrine milieu by culturing them would then allow steroidogenesis to continue. Alternatively, increasing the intrafollicular levels of FSH by treatment may induce IGFBP-4 proteolysis and allow steroidogenesis to proceed. It is impossible to determine whether the follicles have stopped growing because IGFBP-4 levels are higher or vice versa.
IGFBP-4 has a potent IGF-independent antigonadotropic effect on human granulosa cells. No similar effects were seen for IGFBP-2 or -3. These results indicate a physiological reason for the preferential proteolytic cleavage of IGFBP-4 in healthy follicles. The mode of action of IGFBP-4 and the role of IGFBP-2 in granulosa cells are currently under investigation.
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FOOTNOTES |
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1 This work was supported by The Wellcome Trust. 
2 Correspondence. FAX: 44 208 725 2993; rwright{at}sghms.ac.uk
Accepted: April 4, 2002.
Received: November 16, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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activates protein kinase c in human ovarian cells. Mol Cell Endocrinol 1993 91:51-57[CrossRef][Medline]This article has been cited by other articles:
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  M. Li, Y. Li, L. Lu, X. Wang, Q. Gong, and C. Duan Structural, gene expression, and functional analysis of the fugu (Takifugu rubripes) insulin-like growth factor binding protein-4 gene Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2009; 296(3): R558 - R566. [Abstract] [Full Text] [PDF]
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 A. J. Roberts, M. J. Al-Hassan, P. M. Fricke, and S. E. Echternkamp Large variation in steroid concentrations and insulin-like growth factor binding proteins exists among individual small antral follicles collected from within cows at random stages of the estrous cycle J Anim Sci, October 1, 2006; 84(10): 2714 - 2724. [Abstract] [Full Text] [PDF]
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E D Watson, S-E Bae, R Thomassen, S R M Thomson, K Woad, and D G Armstrong Insulin-like growth factors-I and -II and insulin-like growth factor-binding protein-2 in dominant equine follicles during spring transition and the ovulatory season Reproduction, September 1, 2004; 128(3): 321 - 329. [Abstract] [Full Text] [PDF]
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