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Gamete Biology |
a Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic
b Department of Molecular and Cellular Biology, University of California at Berkeley, Berkeley, California 94720
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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cumulus cells, follicle-stimulating hormone, growth factors, oocyte development, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vitro, cumulus expansion can be induced by FSH [5, 6], and in mouse, pig, cattle, and rabbit, also by epidermal growth factor (EGF) [12–16]. The intrinsic tyrosine kinase of EGF-receptor (EGFR) is activated by binding of EGF, resulting in EGFR autophosphorylation and subsequent tyrosine phosphorylation of numerous substrates within the cell [17]. The tyrosine phosphorylation of the EGFR enables interaction with exchange proteins and a downstream activation of several signaling pathways, including mitogen-activated protein (MAP) kinase [18], phosphoinositol 3-kinase [19], STAT [20, 21], and phospholipase C
[22] pathways. So far, it is not known what signaling pathway is involved in regulation of EGF-stimulated cumulus expansion.
Recently, we reported that expansion of cumulus cells in the pig is developmentally regulated; while EGF stimulates expansion of OCCs originating from large antral and preovulatory follicles, OCCs from small follicles (<4 mm) do not expand following EGF treatment [3]. We have also shown that the failure of porcine OCCs from the small follicles to undergo expansion is accompanied by their inability to undergo rearrangement of F-actin and increase production of hyaluronic acid following EGF stimulation [3]. Nevertheless, these OCCs were able to respond to FSH and undergo full expansion accompanied by rearrangement of F-actin and increased secretion of hyaluronic acid. Therefore, we supposed that the failure of porcine OCCs to respond to EGF was caused by the absence or immaturity of EGFR. Alternatively, the absence of response to EGF could result from insufficient development of an EGFR downstream pathway.
Here we report that, while only expansion-capable OCCs from large follicles respond to EGF by a prominent tyrosine phosphorylation of EGFR, the amount of EGFR protein is comparable in OCCs from all follicle sizes. We also provide evidence that pretreatment of OCCs from small follicles by FSH strongly increases their response to EGF both in terms of EGFR tyrosine phosphorylation and OCCs expansion, indicating that the function of EGFR in OCC expansion is FSH dependent.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Culture medium M-199 with Hanks balanced salt solution was purchased from Sevac (Prague, Czech Republic), fetal calf serum (FCS) from Veterinary University in Brno (Czech Republic), culture dishes from Nunclon (Roskilde, Denmark), human recombinant FSH from N.V. Organon (Oss, Netherlands), human recombinant EGF from Genzyme Diagnostics (Russelheim, Germany), polyvinylidene difluoride (PVDF)-membrane Immobilon-P from Millipore (Bedford, MA). We used monoclonal antiphosphotyrosine antibodies PY 20 from Transduction Laboratories (Lexington, KY) and PT 66 from Sigma-Aldrich (Prague, Czech Republic). Rabbit anti-EGFR antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated anti-mouse or anti-rabbit IgG and D-[6-3H] glucosamine hydrochloride were from Amersham (Uppsala, Sweden). The enhanced chemiluminescence (ECL) kit was purchased from Amersham and protein G agarose beads (P 4691) from Sigma-Aldrich. All other listed chemicals were purchased from Sigma-Aldrich.
Isolation and Culture of Oocyte-Cumulus Complexes, Cumulus, and Mural Granulosa Cells
Ovaries of slaughtered gilts were collected at a local abattoir and transported to the laboratory in a thermos. OCCs were released from follicles by aspiration and washed in M-199 supplemented with 6.25 mM Hepes, 20 mM sodium bicarbonate, 0.91 mM sodium pyruvate, 1.62 mM calcium lactate, and antibiotics. OCCs were isolated from 3–4-mm and 6–7-mm follicles, which we will refer to as small and large follicles, respectively.
Only OCCs surrounded by compact multilayered cumulus were selected for experiments; special attention was paid to selecting OCCs with the same size of cumulus within each experimental group. Twenty OCCs were cultured in 1 ml M-199 with 5% fetal calf serum in four-well dishes at 38.5°C, 5% CO2 in air. To stimulate expansion of cumulus cells, the culture medium was supplemented with FSH or EGF at a concentration of 10 ng/ml, as reported previously [3].
To quantify EGFR on different types of cells during follicular growth, we prepared samples with defined numbers of cumulus and mural granulosa cells. For this purpose, we isolated OCCs and pieces of mural granulosa cells (about 300 µm in diameter) from 1–2-, 3–4-, and 6–7-mm follicles by the procedures described above. Cumulus cells, stripped mechanically from 50–100 OCCs, or 100 pieces of mural granulosa cells were vortexed in 0.5 ml of calcium and magnesium-free PBS with 3 mg/ml polyvinylpyrrolidone in a tube for 1 min to prepare the cell suspension. The number of cells in the suspension was determined by hemacytometer and the volume adjusted to contain 5 x 104 cells. The tube was centrifuged at 3000 rpm for 3 min, the excess PBS removed, and samples processed for immunoblotting with EGFR antibodies.
Assessment of Cumulus Expansion
Cumulus expansion was assessed 24 h after the onset of culture using a subjective scoring method [23]. Briefly, no response was scored as 0, minimum observable response was scored as 1, expansion of outer OCCs layers was scored as 2, expansion of all OCCs layers except the corona radiata was scored as 3, and expansion of all OCCs layers was scored as 4.
Production of Hyaluronic Acid by OCCs
Total hyaluronic acid (released in culture medium and retained by OCCs) was measured in this experiment. Groups of 10 OCCs were cultured in 100 µl of the culture medium supplemented with 2.5 µCi of D-[6-3H]glucosamine hydrochloride. The cultures were terminated by adding 10 µl of a solution containing 50 mg/ml pronase 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 3MM filter paper disks. The disks were air dried and then washed three times in 0.5% cetylpyridinium chloride with 10 mM nonradioactive glucosamine hydrochloride for 45 min each. The disks were dried once again, and radioactivity was measured using a liquid scintillation counter.
Immunoblotting
At selected intervals, groups of OCCs or defined numbers of cumulus and mural granulosa cells were lysed in 15 µl of sample buffer for SDS PAGE [24], heated at 100°C for 3 min, and stored at -80°C until use. Proteins of the samples were separated on 7% polyacrylamide gel modified as described [25] and transferred to PVDF membrane. Blots were blocked 1 h with 5% FCS and incubated 2 h with antiphosphotyrosine or anti-EGFR antibody. After incubation with secondary antibodies (1:5000) for 1 h, blots were extensively washed and signal developed by ECL. Following detection of phosphotyrosine, antibodies were stripped in some experiments by incubation of blots in 25 mM TRIS with 2% ß-mercaptoethanol and 0.2% SDS at 70°C for 20 min. The blots were then reprobed with anti-EGFR antibody as described above. The intensity of the bands was analyzed by densitometry using the Advanced Image Data Analyzer software (Raytest Isotopenmessgeraete GmbH, Strawbenhardt, Germany). After detection, the blots were stained by Coomassie blue to assess amount of proteins in each lane.
Immunoprecipitation
Cumulus cells (3 x 105/sample) were lysed in 0.3 ml ice-cold buffer A (50 mM Hepes, pH 7.5, 1% NP-40, 150 mM NaCl, 0.5% sodium deoxycholate, 1 mM sodium vanadate, 1mM 4-(2-aminoethyl) benzensulfonyl fluoride, 1 mM p-nitrofenylphosphate, 0.2 µM aprotinin, 2 µM leupeptin, and 30 µM tosyl-L-phenylalanine) [26] for 25 min. The cell lysate was centrifuged and precleared by incubation with 5 µl protein G agarose beads for 60 min at 4°C. After preclearing, the lysates were centrifuged at 104 rpm and supernatants transferred to new tubes. Antiphosphotyrosine PY 20 was added to cell lysates (2 µl/sample) and incubated for 2 h. The lysates were then supplemented with 5 µl protein G and incubated on a rotator at 4°C overnight. Beads were washed five times with 0.2 ml buffer A, immune complexes were extracted by boiling with 2x concentrated SDS sample buffer, and samples were processed for immunoblotting as described above.
Statistics
Analysis of variance (ANOVA) was used to compare results of densitometry on immunoblots and production of hyaluronic acid. The chi-square test for independence and Fisher exact test were used to analyze data on the expansion of OCCs. The differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunoblotting with EGFR antibodies revealed that OCCs isolated from follicles of all size categories contain similar amounts of 170 kDa EGFR (Fig. 1). In contrast, the amount of EGFR protein in mural granulosa cells decreased by 30% with increasing follicle size. No signal of EGFR was detectable in oocytes. Thus, we concluded that the failure of OCCs from small follicles to expand after EGF treatment reported previously [3] was not caused by the absence or lower numbers of EGFRs in the cumulus cells.
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EGF Induces Tyrosine Phosphorylation of EGFR and Other Cumulus Cell Proteins
EGF treatment of OCCs from large follicles induced robust and reproducible tyrosine phosphorylation of a number of proteins (Fig. 2A, top). Particularly prominent appeared to be the phosphotyrosine content of 42-, 116-, and 170-kDa proteins. As expected, based on the size of pig EGFR [27], the 170-kDa tyrosine phosphorylated protein exactly comigrated with the signal of the EGFR (Fig. 2A, bottom). Immunoprecipitation with antiphosphotyrosine antibody followed by development of blots with EGFR antibody confirmed that the 170-kDa phosphoprotein is indeed identical to EGFR (Fig. 2B). The ability of EGF to stimulate expansion of cumulus cells is therefore likely to be specifically transduced by the action of 170-kDa EGFR that becomes transiently phosphorylated on tyrosine upon activation. This result also suggests that the tyrosine phosphorylation of many other proteins that we have detected is specifically linked to the tyrosine kinase activity of EGFR itself or of kinase(s) regulated by EGFR.
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EGF-Induced Protein Tyrosine Phosphorylation in Cumulus Cells Is Follicle-Size Dependent
Next, we assessed the pattern of tyrosine phosphorylation of the EGFR in cumulus cells isolated from small and large follicles. In cumulus cells from the small follicles, the phosphorylation of the EGFR occurred within 1–3 min, reaching approximately a 2-fold increase over the basal level (Fig. 3, A and B). On the contrary, in cumulus cells from the large follicles, the p170/EGFR tyrosine phosphorylation increased 10–12-fold over the basal level at 10 min after stimulation. In OCCs from both small and large follicles, the EGF-stimulated p170/EGFR tyrosine phosphorylation returned to the basal level within 1 h, with a sharp drop by 30 min after the start of activation (data not shown). Importantly, the low level of EGFR tyrosine phosphorylation correlated with the inability of cumulus cells from small follicles to undergo expansion after EGF stimulation reported previously [3].
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FSH Enhances EGF-Induced Phosphorylation of EGFR
FSH is known to increase binding of EGF to granulosa cells and to induce and maintain EGFR in the follicle [28–31]. We therefore examined what the effect of FSH is on the pattern of tyrosine phosphorylation of EGFR in cumulus cells of small follicles. OCCs from small follicles were cultured in medium with or without FSH for 3 h and were stimulated by EGF afterward. In control groups preincubated without FSH, the phosphorylation of EGFR increased about 2-fold over the base level. However, in the group of OCCs preincubated with FSH, the EGF-induced tyrosine phosphorylation of EGFR was about 9-fold over the base level, and the overall pattern of tyrosine phosphorylation resembled EGF-treated OCCs from large follicles (Fig. 3, C and D). FSH itself did not affect tyrosine phosphorylation of EGFR (Fig. 3C). In summary, these results suggest that FSH treatment in vitro is sufficient to induce development of a mature EGF response in cumulus cells of small follicles. This effect of FSH was not caused by increasing numbers of EGFRs on cumulus cells during the time of preincubation (Fig. 4).
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FSH-Enhanced Phosphorylation of EGFR Promotes Expansion of OCCs
In the next experiment, we asked whether sequential treatment by FSH and EGF promotes production of hyaluronic acid and expansion of OCCs from small follicles. As expected, based on our previous results [3], OCCs from small follicles did not expand following stimulation by EGF alone, irrespective of the length of stimulation (1 or 24 h; Fig. 5A). In contrast, more than 85% of OCCs cultured in FSH-supplemented medium for 24 h underwent expansion. Thirty percent of OCCs cultured in FSH for 4 h and subsequently in control medium for 20 h underwent expansion, while sequential treatment with FSH (3 h) followed by EGF (1 h) and control medium (20 h) induced expansion in 53% of OCCs (P < 0.05). In concert with these data, production of hyaluronic acid was significantly higher in OCCs stimulated by the sequential treatment than in OCCs stimulated by FSH or EGF (Fig. 5B). The FSH-dependent development of a mature level of EGF-induced EGFR phosphorylation (Fig. 3C) is therefore strongly correlated with increased ability of OCCs to produce hyaluronic acid and to undergo expansion.
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EGFR Tyrosine Kinase Activity Is Essential for EGF-Induced Expansion of OCCs
The results of the above experiments suggest that activity of the intrinsic EGFR-tyrosine kinase is involved in regulation of EGF-induced expansion. To confirm this, we assessed the effect of EGFR-tyrosine kinase-specific inhibitor tyrphostin 46 [32] on expansion of porcine OCCs from large follicles. As shown in Figure 6, tyrphostin inhibited expansion in a dose-dependent manner, with full inhibition at a concentration of 10 µM.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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EGF binding to its receptor causes activation of the intrinsic receptor tyrosine kinase, which results in autophosphorylation of the EGFR [17]. Tyrosine kinase activity of EGFR is essential for activation of the downstream signaling pathways [18, 20, 34, 35], regulation of gene expression [36, 37], cell proliferation [35], and apoptosis [38]. Also, effects of EGF on regulation of bovine oocyte maturation [39] and EGF-induced production of hyaluronic acid by mouse OCCs [40] are mediated through a tyrosine kinase pathway. Similarly, we found in the present study that tyrphostin 46 is able to eliminate EGF-induced expansion of porcine OCCs, providing evidence that tyrosine kinase activity is required in this process.
We found three cumulus cell proteins heavily phosphorylated on tyrosine residues after stimulation with EGF. First was a protein of 170 kDa, which showed the highest increase in phosphorylation and which we identified in the next experiments as the EGFR; second was a protein of 116 kDa that showed a similar pattern of phosphorylation as the EGFR. We assume that this protein may be the product of the Cbl proto-oncogene that was also identified as a 116-kDa tyrosine phosphorylated protein in a variety of EGF-stimulated cells [41, 42]. Finally, the 42-kDa protein might be identical to the MAP kinase because it comigrated on one-dimensional immunoblots with activated ERK-2 protein (data not shown).
The comparison of tyrosine phosphorylation of EGFR localized on small and large follicles indicates that the extent of EGFR phosphorylation may play a pivotal role in regulation of EGF-stimulated expansion of OCCs. Only expanding cumulus cells from large follicles contained EGFR capable of extensive tyrosine phosphorylation following stimulation by EGF. In contrast, EGFR in nonexpanding cumulus cells from small follicles exhibited only low levels of EGF-induced tyrosine phosphorylation. There are several possible explanations of this feature.
OCCs from small follicles may contain EGFR with a decreased binding of EGF. To our knowledge, no data on EGF binding capacity of cumulus cells during folliculogenesis have been reported so far. In mural granulosa cells, the EGFR binding capacity was primarily affected by their luteinization and decreased with follicle enlargement due to a decrease in receptor number with no change in receptor affinity [28]. However, it is unlikely that a similar scenario would apply in the case of cumulus cells that are under the influence of oocyte-secreted paracrine factors preventing them from luteinization [23]. Indeed, our immunoblotting data revealed that EGFR concentration decreases in mural granulosa cells and remains stable in cumulus cells during follicular growth.
Although all EGFRs in EGFR-expressing cells are molecularly identical, they can be divided into two classes that have either low or high affinity to EGF [43]. The mechanisms responsible for transmodulation of EGFR have been extensively studied but remain still unclear. The increase in the fraction of high-affinity EGFR, associated with a high level of EGF-induced autophosphorylation, was attributed to binding of the EGFR to filamentous actin [44]. However, removal of the actin-binding site from the receptor did not affect the affinity of EGFR to EGF [43]. Next, EGFR can form homodimers or heterodimers with other members of the EGFR family, including ErbB2 [45]. These dimeric forms of EGFR were shown to bind EGF with high affinity [46, 47]. On the other hand, substances that disrupt high-affinity EGF-EGFR interaction in HeLa cells had no effect on the EGFR homo- or heterodimerization [48]. Instead, the presence of specific EGFR-affinity modulating proteins has been suggested [43, 48]. This idea is supported by the discovery of a peptide that induces conformational change of the EGFR and thus provides access to additional tyrosine autophosphorylation sites [49]. In addition, tyrosine kinase activity of the EGFR was enhanced 10-fold after binding with the oncogenic form of c-Cbl in EGF-stimulated lymphoma cells, suggesting that Cbl protein acts as a regulator of receptor tyrosine kinases [50]. We propose that one of these mechanisms may be responsible for the differing extents of phosphorylation of EGFR in cumulus cells from small and large follicles. It should be noted at this point that, in our experiments, the extensive phosphorylation of EGFR was regularly accompanied by increased tyrosine phosphorylation of the 116-kDa protein, which, as discussed above, may be identical to the Cbl.
Gonadotropins and EGF have a synergistic effect on the range of functions of follicular cells. FSH was shown to increase the number of EGFRs and binding of EGF on cultured granulosa cells in a dose-dependent manner [28–31, 51]. In our experiment, we demonstrated that FSH promotes maturation of the EGF-response pathway in OCCs from small follicles, as evidenced by a strong increase in EGF-induced EGFR tyrosine phosphorylation, production of hyaluronic acid, and OCCs expansion. Surprisingly, a 3-h preincubation of OCCs in FSH-supplemented medium was sufficient to induce a significant increase in EGFR phosphorylation without an increase in EGFR concentration. These data indicate that FSH may be implicated in mechanism(s) regulating EGFR tyrosine phosphorylation in porcine cumulus cells. We suggest that FSH-dependent maturation of EGFR may also function in vivo and regulate expansion of cumulus cells in preovulatory follicles.
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FOOTNOTES |
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2 Correspondence: Radek Prochazka, Academy of Sciences of the Czech Republic, Institute of Animal Physiology and Genetics, Rumburska 89, 277 21 Libechov, Czech Republic. FAX: 420 315 69 71 86; e-mail: prochazka{at}iapg.cas.cz
Received: 12 April 2002.
First decision: 6 May 2002.
Accepted: 10 September 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inhibitor binding to hyaluronan in the cumulus extracellular matrix is required for optimal ovulation and development of mouse oocytes. Biol Reprod 1999 61:436-443This article has been cited by other articles:
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