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Articles |
a Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Lib
chov, Czech Republic
b Ottawa Regional Cancer Centre and Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada K1H 8M5
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cumulus expansion in vitro normally requires the presence of serum; however, Singh and Armstrong [10] observed that the addition of exogenous insulin-like growth factor-I (IGF-I) to a serum-free medium enabled cumulus cells to expand in response to FSH. In the human, it has been reported that serum contains high concentrations of IGF-I (normal range in an adult is ~200 ng/ml; during puberty, concentrations rise to levels 2–3 times those seen in adults) [11]. Singh and Armstrong [10] suggested that IGF-I is a component of serum that enables cumulus cells to expand in response to FSH in vitro. Since IGF-I is present in the follicle in vivo, it may have a physiological role during gonadotropin-induced cumulus expansion.
In response to the preovulatory gonadotropin surge, cumulus granulosa cells secrete hyaluronidase-sensitive mucous materials: proteoglycans and glycosaminoglycans. Proteoglycans are large macromolecules consisting of numerous heteropolysaccharide side chains called glycosaminoglycans, covalently linked to a protein core. Their biosynthesis is regulated by FSH, prostaglandin E2, and transforming growth factor ß1. Proteoglycans found in follicular fluid or secreted by granulosa in vitro contain predominantly chondroitin sulfate. In contrast, the major structural macromolecule in the matrix of the expanded cumulus cells is HA, indicating a metabolic divergence in glycosaminoglycans between granulosa cells and cumuli [12]. In mouse complexes, net synthesis of HA during FSH-stimulated expansion of the oocyte-cumulus complex (OCC) correlates directly with accumulation of glycosaminoglycan in the matrix and with the morphological changes associated with expansion of the OCC [2, 13]. Moreover, treatment of FSH-stimulated mouse complexes with Streptomyces hyaluronidase indicated that 90% of the [3H]glucosamine incorporated into cetylpyridinium hydrochloride-precipitable counts was incorporated specifically into HA [13]. In the presence of fetal bovine serum, the HA was retained within the complexes, but in the absence of fetal bovine serum it was released into the culture medium. Retention of HA within the complex was correlated with cumulus expansion or mucification [14].
The role of the oocyte in porcine cumulus expansion and HA synthesis is still not clear. Nakayama et al. [15] found that HA synthesis by cumulus cells during cumulus expansion was dependent on the oocyte, since oocytectomy reduced this HA synthesis. According to their results, porcine follicular fluid induces cumulus expansion in intact and OOX complexes cultured for over 24 h; but the degree of expansion in OOX reached only 76% that of intact complexes, suggesting that cumulus expansion by porcine OCC is regulated by oocyte-dependent mechanisms. In contrast, when FSH is used to induce cumulus expansion, we have previously found no difference between intact and OOX porcine complexes in their degree of expansion; however, the intracellular cAMP content in intact complexes was significantly higher than in OOX [8]. It is likely that the different mechanisms used to stimulate cumulus expansion may account for the ability to identify different oocyte-dependent responses. However, analysis of the results may be further complicated by the fact that both studies evaluated the expansion and/or HA content in complexes alone, which may not accurately reflect the full HA synthetic capacity of the cumulus cells. Therefore our present work addressed the following aims: 1) to determine, using oocytectomy, whether FSH-stimulated synthesis and retention of HA by porcine OCC are dependent upon the oocyte and/or are influenced by the presence of serum and 2) to determine the effects of IGF-I on the ability of cumulus cells to synthesize and retain HA in response to FSH.
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MATERIALS AND METHODS |
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Porcine ovaries were obtained from crossbred gilts (Landrace, Large White) at a local abattoir and transferred to the laboratory in a thermos at 30°C. Medium-sized antral follicles were aspirated. Only complexes containing fully grown germinal vesicle breakdown-competent oocytes (diameter 120 µm) isolated from 3- to 5-mm follicles were selected. The culture medium was M-199 (Sevac, Prague, Czech Republic) buffered with 20 mM NaHCO3 and 6.25 mM Hepes and supplemented with 5% fetal calf serum (FCS), 0.91 mM sodium pyruvate, 1.62 mM calcium lactate, and antibiotics [16]. In the absence of serum, polyvinylpyrrolidone (PVP, 3 mg/ml; Sigma Chemical Co., St. Louis, MO) was added into the culture medium. Porcine FSH (1 µg/ml, iodination grade; Biogenesis, Poole, UK) and IGF-I (100 ng/ml; Pharmacia Diagnostics AB, Bromma, Sweden) were used for these experiments.
Oocytectomy
Some of the porcine OCC were deprived of the oocyte (oocytectomized) by the method described previously [1, 8]. Each OCC was attached to a holding pipette. A glass needle was then introduced through the cumulus cells and the oocyte into the holding pipette. Upon withdrawal of the needle, the ooplasm was aspirated into the holding pipette by a burst of negative pressure. The resulting OOX was thus composed of an evacuated zona and surrounding cumulus cells.
Assessment of Cumulus Expansion
Ten OCC or OOX were added to 100 µl of culture medium, and their expansion was assessed 24 h later according to the subjective scoring system (0 to +4) described previously [8]. Briefly, 0 indicates no detectable response; +1 indicates the minimum observable response: peripheral cumulus cells with a glistening appearance; +2 indicates expansion of the peripheral layers of the cumulus; +3 indicates expansion of all layers of the cumulus except corona radiata cells; and +4 indicates expansion of the cumulus including corona radiata cells.
HA Synthesis
Groups of 10 porcine OCC or OOX were cultured for 24 h at 38°C in an atmosphere of 5% CO2 in air and in the presence of 2.5 µCi of D-[6-3H]glucosamine hydrochloride (Amersham, Arlington Heights, IL) and 1 µg/ml FSH. HA synthesis was measured using a procedure described by Eppig [14, 17] with slight modifications. Briefly, the cultures were terminated by adding 10 µl of a solution containing 50 mg/ml pronase (Sigma) and 10% Triton X-100 in 0.2 M Tris buffer, pH 7.8. The samples were incubated for 2 h at 38°C and then transferred to Whatman (Clifton, NJ) 3MM filter paper circles. The circles were air dried and then washed through three changes of solution containing 0.5% cetylpyridinium chloride and 10 mM nonradioactive glucosamine hydrochloride (Sigma) for 45 min each. The circles were dried once again, and radioactivity was measured using a liquid scintillation counter. Synthesis of HA was measured either in medium plus OCC or OOX (total HA) or within the complexes alone (retained HA); this was achieved by simply transferring the complexes through three changes in culture medium without labeled precursor before addition of the pronase-Triton X-100 solution. The specificity of incorporation of radioactivity into HA was determined by sensitivity to highly specific Streptomyces hyaluronidase (Sigma). After the overnight culture period, some samples were treated with 10 IU Streptomyces hyaluronidase for 2 h at 38°C prior to addition of the pronase-Triton X-100 solution.
Statistical Analysis
Differences between responses to the various treatments were identified by ANOVA, followed by Bonferroni's test to determine significance between specific treatment groups or unpaired, two-tailed t-tests when only two treatments were being compared. For all figures, error bars indicate the SEM from at least five independent experiments, signifying that at least 50 complexes (OCC or OOX) were used per treatment.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At the end of the culture period, intact and OOX complexes were assessed microscopically for expansion according to a subjective scoring system [8]. FSH stimulated full expansion (+3 to +4) of both intact OCC and OOX after 24-h culture in medium supplemented with 5% FCS (Fig. 1). Treatment of the complexes with Streptomyces hyaluronidase caused the mucified cumulus of both OCC and OOX to disperse, indicating that HA was an integral component of expanded cumulus (Fig. 1).
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FSH-Stimulated HA Synthesis by OCC and OOX
Treatment of intact OCC with FSH and FCS resulted in a significant increase in the synthesis of HA compared to that in complexes cultured in the absence of FSH (Fig. 2; p < 0.001). Oocytectomy did not influence the cumulus expansion, such that the total HA accumulation by OOX was not different from that of intact OCC (Fig. 2; p > 0.05). Moreover, there were no differences in the amount of HA retained within the complexes between these groups (Fig. 2; p > 0.05). For both intact and OOX complexes, the amount of HA retained within the complexes represented about 55% of the total amount accumulated in the well (complexes + medium). More than 90% of the total incorporated radioactivity was sensitive to highly specific Streptomyces hyaluronidase, indicating that at least 90% of the incorporated [3H]glucosamine was in HA (Fig. 2).
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Role of Serum in Synthesis of HA
HA synthesis by intact OCC was measured in the presence or absence of 5% FCS. In the presence of FCS, highly purified FSH stimulated cumulus expansion (+3 to +4) and synthesis of HA (Fig. 3). In the absence of FCS, when PVP (3 mg/ml) was added to the culture medium, total HA accumulation was reduced by 35% (p < 0.01). In addition, when PVP replaced FCS, the retention of HA within the complexes decreased to less than 40% of the amount retained within the complexes when they were cultured in the presence of serum (p < 0.01).
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Role of Oocyte in the Synthesis of HA in Serum-Free Medium
To investigate the role of oocytes in synthesis of HA in serum-free medium, total and retained HA synthesis by intact and OOX complexes was measured in medium supplemented with PVP. As shown in the previous experiment, FSH stimulated synthesis of HA by intact complexes, and the amount retained was significantly less than the total HA accumulated in the well (Fig. 4). There were no differences between intact and OOX complexes in their ability to synthesize HA or in the amount retained within the complexes (p > 0.05), suggesting that, in PVP-supplemented medium, FSH induced HA synthesis independently of the paracrine factor from the oocyte.
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Effect of IGF-I on Synthesis of HA
To examine the effects of IGF-I on HA synthesis, intact OCC were cultured in medium supplemented with 5% FCS, PVP (3 mg/ml) and IGF-I (100 ng/ml), or PVP (3 mg/ml) alone. In all cases, FSH was added to induce cumulus expansion. The presence of IGF-I enabled cumulus cells to expand (+3 to +4) and to synthesize HA in response to FSH in serum-free medium (Fig. 5). Indeed, the total accumulation of HA in wells containing IGF-I-treated complexes was comparable to that observed when serum was present in the culture medium, suggesting that IGF-I may be the serum-derived factor that enhances HA production by cumulus cells. Moreover, the amount of HA retained within the complexes was not significantly different between these two groups (p > 0.05, Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Species differences have already been documented for the role of the oocyte in the regulation of cumulus cell expansion [4, 8]. Buccione et al. [1] demonstrated that mouse oocytes secrete a CEEF that allows the cumulus cells to synthesize HA and undergo cumulus expansion in response to FSH and epidermal growth factor. Moreover, the results of Tirone at al. [18] indicated that the oocyte factor(s) must be continually present for maximum cumulus expansion and HA synthesis. Porcine oocytes secrete CEEF; however, in contrast to what occurs in mice, both the cumulus and mural granulosa of porcine follicles are also capable of producing CEEF [9]. Therefore, both paracrine secretion of CEEF by oocytes and mural granulosa cells and the observed autocrine secretion of CEEF by cumulus cells themselves are involved in cumulus expansion in porcine follicles.
The requirement for the oocyte in promoting porcine cumulus expansion and HA synthesis is still unclear. Nakayama et al. [15] found that follicular fluid-induced expansion of porcine OCC was reduced by oocytectomy; however, in this and a previous study [8], we never observed differences in cumulus expansion between intact and OOX complexes. The differences in results most likely reflect utilization of FSH for inducing cumulus expansion in our experiments instead of follicular fluid, and would suggest that, in contrast to follicular fluid, FSH is capable of eliciting a response(s) in cumulus cells that enables full HA synthetic capacity in an oocyte-independent manner.
The difference between the expansion-inducing abilities of follicular fluid and FSH seems to lie specifically in their abilities to stimulate HA synthesis. Suchanek et al. [12] reported that proteoglycans found in follicular fluid contain predominantly chondroitin sulfate. In contrast, cumulus cells secrete HA. Net synthesis of HA during FSH-stimulated expansion of the OCC correlates directly with accumulation of glycosaminoglycan in the matrix [2, 13]. In this regard, our results are in agreement with those of Nakayama et al. [15] in that both follicular fluid- and FSH-stimulated incorporation of [3H]glucosamine into porcine OCC were minimally affected by oocytectomy. However, it appears that follicular fluid and FSH have divergent effects on inducing the ability of OOX to incorporate the precursor into glycosaminoglycans. Nakayama et al. [15] demonstrated that OOX were deficient in their ability to generate HA, but were fully capable of utilizing substrate for chondroitin sulfate synthesis. In contrast, this study shows that FSH stimulated levels of HA synthesis in OOX that were equivalent to those in OCC. It is clear that, at least in pigs, FSH is required to ensure production of the appropriate glycosaminoglycan during cumulus expansion.
While we have found that the degree of cumulus expansion induced by FSH appears to be similar in intact and OOX porcine complexes, the ability to generate cAMP was significantly different, such that intact OCC had significantly higher cAMP content than their oocytectomized counterparts [8]. We originally hypothesized that the differing levels of cAMP may reflect differing abilities to synthesize or retain HA; however, our results indicate that intact and OOX complexes have equal capacities to synthesize and retain HA, despite different cAMP contents. This is in dramatic contrast with what occurs in mouse complexes, where neither expansion nor HA synthesis occurs in response to FSH or cAMP analogues in the absence of mouse oocytes, despite the fact that FSH stimulates similar levels of cAMP generation in intact and OOX complexes [1, 2]. More recent results have demonstrated that FSH-stimulated intracellular cAMP synthesis correlates with net HA production up to 14 fmol/OCC at 5 ng/ml FSH; however, higher concentrations of FSH increased cAMP levels 10-fold with no additional effect on HA synthesis [18]. This may explain why different levels of cAMP produced by FSH-stimulated porcine OCC and OOX do not affect the level of HA synthesis.
It has previously been demonstrated that serum plays a critical role in mouse cumulus expansion by promoting the retention of HA synthesized de novo in response to FSH [14]. Our results from experiments in which serum was replaced with PVP suggest that porcine cumulus cells are still capable of responding to FSH and synthesizing HA—but that the levels generated are reduced, both in total and with respect to retention within the complexes. In addition, there was a significant decrease in the proportion of total HA synthesis retained within the complexes compared with that in cultures supplemented with FCS. These results are in agreement with earlier studies with mouse complexes, which showed that in the presence of serum, HA was retained within the complexes, but in the absence of serum it was released into the culture medium [14, 19].
The addition of IGF-I to the chemically defined medium enabled cumulus cells to expand in response to FSH in a manner similar to that observed when serum was present in the culture medium. These results are in agreement with the observations of Singh and Armstrong [10], who suggested that IGF-I is a component of serum that enables cumulus cells to expand in response to FSH in vitro and that the effect is receptor mediated. We have extended these observations further by demonstrating that the ability of IGF-I to enhance cumulus expansion in chemically defined media is associated with normal capabilities of the cumulus cells to both synthesize HA and retain HA within the complex. The ability of such defined media to support normal HA synthesis and cumulus expansion will enable investigators to characterize more precisely the CEEF produced in porcine follicles.
We conclude that the synthesis of HA by porcine cumulus cells in vitro is stimulated with FSH and that oocytectomy changes neither the total synthesis nor the retention of HA within the complexes in medium supplemented with serum. Moreover, our results indicate that FSH-stimulated full expansion of the cumulus and synthesis of HA can be accomplished in a chemically defined medium containing IGF-I.
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ACKNOWLEDGMENTS |
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. Hladk
and R. Mocová for excellent technical assistance. |
FOOTNOTES |
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2 Correspondence. FAX: 420 206 697 186; nagyova{at}iapg.cas.cz
Accepted: April 8, 1999.
Received: February 9, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ová Z, Nagai T, Kikuchi K. Motlík J. Lack of effect of oocytectomy on expansion of the porcine cumulus. J Reprod Fertil 1991; 93:569–576.This article has been cited by other articles:
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