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Ovary |
-Trypsin Inhibitor Family Proteins to Hyaluronan in In Vivo and In Vitro Expanded Porcine Oocyte-Cumulus Complexes1
Institute of Animal Physiology and Genetics,3 Libechov, Czech Republic
Department of Public Health and Cell Biology,4 Faculty of Medicine, University of Rome, Rome, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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I) molecules become covalently linked to hyaluronan (HA) during in vivo mouse cumulus expansion and significantly contribute to cumulus matrix organization. Experiments with mice suggest that the incorporation of such proteins in cumulus matrix appears to be rather complex, involving LH/hCG-induced changes in blood-follicle barrier and functional cooperation between cumulus cells, granulosa cells, and oocyte within the follicle. We demonstrate here that HC-HA covalent complexes are formed during in vivo porcine cumulus expansion as well. Western blot analysis with II antibody revealed that follicular fluids from medium-sized follicles and those from large follicles unstimulated with hCG contain high levels of all forms of II family members present in pig serum. The same amount of HCs were covalently transferred from II molecules to HA when pig oocyte-cumulus complexes (OCCs) were stimulated in vitro with FSH in the presence of pig serum or follicular fluid from unstimulated or hCG-stimulated follicles. In addition, HC-HA coupling activity was stimulated in cumulus cells by FSH treatment also in the absence of oocyte. Collectively, these results indicate that II molecules can freely cross the blood follicle barrier and that follicular fluid collected at any stage of folliculogenesis can be successfully used instead of serum for improving OCC maturation. Finally, pig cumulus cells show an autonomous ability to promote the incorporation of II HCs in the cumulus matrix.
cumulus cells, fertilization, follicle-stimulating hormone, ovary, ovulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence indicate that serum components belonging to inter-alpha-trypsin inhibitor family (II) play a key role in cumulus matrix formation. Mouse oocyte-cumulus complexes (OCCs) induced to expand in vitro by FSH require the presence of serum to retain the newly synthesized HA in the matrix [4–6]. Organization of HA-enriched matrix of the cumulus does not occur with II-immunodepleted serum while it does in the presence of purified II molecules [7]. Finally, mice lacking intact II family members fail to form a stable cumulus matrix and the naked ovulated oocytes are not fertilized in vivo [1].
These molecules consist of a small protein, named bikunin or light chain, with a chondroitin sulfate moiety that contains one or two evolutionarily related proteins, named heavy chains (HC1, HC2, HC3). II carries two HCs, HC1 and HC2, while pre--inhibitor (PI) and inter--like inhibitor (ILI) have one HC, HC3 and HC2, respectively [8]. II-related molecules are assembled in the Golgi apparatus of hepatocytes and circulate in the blood. It has been reported that these serum molecules are excluded from mouse ovarian follicle until an LH/hCG ovulatory stimulus is provided [9, 10], implying that hormonal control of blood-follicle barrier might be critical for successful cumulus expansion.
When inside the follicle, the HCs are transferred from the glycosaminoglycan of II-related molecules to HA produced by cumulus cells through a transesterification process [11]. It has been proposed that the covalent linkage of HCs to HA is critical for cross-linking HA strands and stabilizing cumulus matrix. The kinetics of HC-HA complex formation by mixing purified II and HA with follicular fluid indicates that factors produced within the follicle after LH/ hCG administration catalyze this reaction [12]. Indeed, it has been shown that tumor necrosis factor-induced protein-6 (TNFIP6; also known as tumor necrosis factor stimulated gene-6, TSG-6), a protein with the ability to bind HA and II molecules, is produced by cumulus and granulosa cells after an ovulatory stimulus [13, 14], in parallel to HA synthesis by OCCs [5]. More important, covalent transfer of HCs to HA does not occur in OCCs of Tnfip6 null mice, indicating that TNFIP6 is critically involved in this process [2]. In agreement with the essential role of oocyte in promoting cumulus expansion in mouse [15, 16], TNFIP6 production in the mouse follicle appears to require the paracrine stimulus by an oocyte factor, likely growth differentiation factor 9 [17].
Regulation of porcine and bovine cumulus expansion appears to differ in some aspects from that of mouse cumulus expansion. For instance, although porcine and bovine oocytes produce soluble factors enabling HA synthesis and expanded matrix formation by FSH-stimulated mouse oocytectomized complexes (OOXs) [18–21], pig and bovine FSH-stimulated cumulus cells do not require the presence of oocytes to synthesize HA and undergo expansion in vitro [20, 22, 23]. Moreover, we have shown previously that porcine cumulus cells were able to expand, synthesize, and retain HA after FSH stimulation in the absence of serum. Nevertheless, there was a significant decrease in the proportion of HA retained within the complexes compared with that in cultures supplemented with serum [23].
Despite the importance assigned to HC incorporation in cumulus matrix and fertility, no study has been performed on the occurrence and regulation of this process in species other than mouse [1, 7]. Therefore, we have investigated whether HCs are covalently linked to HA in pig OCCs expanded in vivo and in vitro in the presence of serum. In addition, to investigate the role of follicle environment in regulating this reaction, pig follicular fluid collected before and after hCG injection was analyzed for II molecule content and for ability to promote or enhance HC covalent linkage to HA in porcine OCCs stimulated in vitro with FSH. The role of oocyte in regulating this process has also been investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To examine in vivo unexpanded and expanded OCCs, gilts from F1 crosses between Minnesota and Gotingen strains of miniature pigs were stimulated with 500 IU eCG (Folligon, Intervet, The Netherlands) on Day 15 of the cycle. Animals were slaughtered either 72 h after eCG stimulation or 24 h after a subsequent injection of 500 IU hCG (Organon, Oss, The Netherlands). OCCs were isolated from ovaries by aspiration of large preovulatory follicles about 10 mm in diameter both after eCG stimulation (unexpanded OCC) and after hCG injection (expanded OCC).
Unexpanded porcine OCCs for in vitro culture were obtained from ovaries of crossbred prepubertal gilts (Landrace, Large white) at a local abattoir. The complexes were collected by aspiration of medium-sized antral follicles about 3–6 mm in diameter. The Animal Research Committee of Academy of Sciences of the Czech Republic approved all procedures.
Oocytectomy
Some of the porcine OCCs were deprived of the oocyte (oocytectomized) by the method described previously [16, 22]. 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 oocytectomized complexes (OOXs) were thus composed of an evacuated zona and surrounding cumulus cells.
Culture of OCCs and OOXs
After isolation, OCCs and OOXs were transferred to 300 µl of culture medium in four-well plates. Culture medium was M-199 (Sevac, Prague, Czech Republic) supplemented with 20 mM NaHCO3, 6.25 mM HEPES, 0.91 mM sodium pyruvate, 1.62 mM calcium lactate, antibiotics, and containing 100 ng/ml recombinant FSH (Organon). Ten percent pig serum or 10% fetal bovine serum (both from Sigma-Aldrich, Prague, Czech Republic), or 10% porcine follicular fluid was added into the culture medium. The follicular fluid was obtained from antral follicles or from follicles after 24 h of hCG injection. The complexes were cultured for 24–28 h at 38.5°C in an atmosphere of 5% CO2 in air.
Collection of Follicular Fluid
Follicular fluid was aspirated from antral follicles (about 5 mm in diameter) of ovaries from a slaughterhouse or from preovulatory follicles obtained from superovulated cycling gilts. The eCG-stimulated gilts were killed either 72 h after eCG injection or 8 and 24 h after the subsequent injection of hCG. The preovulatory follicles were aspirated and the follicular fluids obtained from one animal were pooled. In all cases, follicular fluids were centrifuged for 20 min at 1500 x g, and the supernatants were stored at –20°C.
Western Blot Analysis
Complexes obtained after in vivo stimulation or after in vitro culture were washed three times in PBS containing a cocktail of protease inhibitors (PBS-PI) (Complete; Mini, Boeringher Mannheim, Mannheim, Germany). They were then either directly solubilized in reducing Laemmli buffer, containing 2% sodium dodecyl sulfate (SDS) and 5% ß-mercaptoethanol, or digested with 1 IU of Streptomyces hyaluronidase (Merck-Calbiochem, Prague, Czech Republic) in 20–30 µl of PBS-PI at 37°C for 2 h before adding 5x concentrated reducing Laemmli buffer, to allow proteins covalently linked to HA to enter the gel. In some experiments, the digested samples were centrifuged at 300 x g for 5 min to separate the matrix extract from the cell pellet. Reducing Laemmli buffer was then added to each fraction.
Pig serum or follicular fluid (4 µl) were either directly solubilized in reducing Laemmli buffer or digested with Streptomyces hyaluronidase (1 IU) or with chondroitinase ABC (50 mU) (Merck-Calbiochem) in PBS-PI (20 µl) at 37°C for 2 h before adding 5x concentrated reducing Laemmli buffer.
All samples were then boiled at 100°C for 4 min, separated in 7.5% acrylamide/SDS gels, and transferred to Hybond-P membranes (Amersham-Pharmacia). Membranes were blocked with 5% nonfat dry milk in TBS for 2 h at room temperature. Primary and secondary antibodies were diluted in TBS containing 5% BSA and 0.05% Tween 20. Proteins of the II family were detected using rabbit anti-human II antibody (1:2000, DAKO, Carpenteria, CA) incubated overnight at 4°C, followed by horseradish peroxidase-labeled anti-rabbit immunoglobulin (1:2000, Amersham-Pharmacia) as secondary antibody for 1 h at room temperature. After primary and secondary antibody, 2 x 5 min and 3 x 10 min washes were made in TBS containing 0.1% Tween 20. The positive reaction was detected by enhanced chemiluminescence (Amersham-Pharmacia).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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I-Related Molecules and HA in Porcine OCCs Expanded In Vivo
To determine whether HCs of serum-derived II-related molecules are covalently linked to HA in pig cumuli expanded in vivo, OCCs were isolated from antral follicles of pigs treated with eCG (unexpanded OCCs) or eCG followed by hCG for 24 h (expanded OCCs; Fig. 1A). The results show that unexpanded porcine OCCs either undigested (data not shown) or digested (Fig. 2, lane 1) with hyaluronidase did not contain any protein reactive with II antibody. Conversely, expanded OCCs contained positive bands of about 220, 130, and 120 kDa that were detected also in porcine serum (Fig. 3A, lane 1), likely corresponding to II (bikunin plus HC1 and HC2), PI (bikunin plus HC3), and ILI (bikunin plus HC2), respectively [24, 25]. After digestion with hyaluronidase, two additional immunopositive bands of about 75–85 kDa and 95 kDa were detected in the extract of OCCs, the former likely corresponding to the relative molecular mass of single HC1 and HC2 and the latter to that of a single HC3 [8, 26]. These results indicate that HCs from each II-related molecules identified in the pig serum were transferred and covalently linked to HA during in vivo pig cumulus expansion. The two minor positive bands in the hyaluronidase extract of expanded OCCs migrating at about 160–170 kDa likely represent dimers of the different types of HCs linked to the same HA strand at so close a proximity that the enzymatic digestion of the interposed HA fragment is prevented, as previously suggested [11]. Analysis of the matrix and cell extracts (cumulus cells plus oocytes) separately confirmed that the immunoreactivity was exclusively associated with the matrix.
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Analysis of II-Related Molecules in Porcine Follicular Fluid
It has been reported that serum II-related molecules are excluded from mouse antral follicles until an ovulatory dose of LH or hCG changes the permeability of the blood-follicle barrier [9, 10]. This observation is apparently in contrast with the ability of pig follicular fluid from medium-sized antral follicles to support full expansion of in vitro hormone-stimulated OCCs in the absence of serum [27]. To clarify this discrepancy, porcine follicular fluids were collected at different stages of folliculogenesis and analyzed for the presence of II proteins. Results reported in Figure 3A show three major bands recognized by the II antibody in follicular fluid aspirated from medium-sized follicles that migrate at the relative position of II, PI, ILI present in porcine serum. The identification of these proteins as II family members is supported by the evidence that they disappear in follicular fluid digested with chondroitinase and give rise to proteins migrating at 95 and 75–85 kDa (Fig. 3B, lane 3), corresponding to the molecular weight of free HC3 and HC1/HC2, respectively. The residual positivity at about 170 kDa in chondroitinase-treated follicular fluid was previously detected in immunoblotting of purified II treated with chondroitinase as well. It represents a small fraction of HC1 and HC2 likely linked by a chondroitinase-resistant glycosaminoglycan bond [24]. Digestion of follicular fluid with Streptomyces hyaluronidase had no effect on the immunoreactive profile, indicating that this enzyme preparation was free of chondroitinase contaminant (Fig. 3B, lane 2).
The levels of II molecules in pig follicular fluid changed neither in eCG-primed follicles nor in 8-h hCG-stimulated follicles, while a detectable increase of concentration was observed at 24 h post-hCG injection (Fig. 3C).
Collectively, these results indicate that there is no apparent barrier to the transfer of II family molecules from the blood to the follicle in pig and that LH/hCG only facilitates their diffusion.
HCs Are Covalently Transferred to HA During In Vitro OCC Expansion
Previous studies have shown that factors synthesized within the follicle catalyze the covalent transfer of HCs from II to HA [2, 11, 12]. To investigate whether porcine OCCs have the ability to promote this reaction, unexpanded OCCs were isolated from the follicles and induced to expand in vitro with FSH in the presence of pig serum or pig follicular fluid collected from medium size follicles (Fig. 1B). Western blot analysis with II antibody of protein extracts of OCCs cultured in both conditions revealed a positive 75- to 85-kDa material, corresponding to free HCs, that was fully associated with the matrix and released after hyaluronidase digestion, as in OCCs expanded in vivo (Fig. 4, A and C). Similar results were obtained by stimulating OCCs in the presence of follicular fluid collected 24 h after hCG (Fig. 4B). The small amount of immunopositive material corresponding to the native II molecules in the total hyaluronidase extracts (220, 130, and 120 kDa) suggests that the conversion of these molecules to HA-linked HCs by OCCs is almost complete in these culture conditions. Interestingly, when fetal bovine serum was used as a source of II molecules, no protein could be detected by the anti-human II antibody neither in the matrix nor in the cell extracts of FSH-stimulated OCCs (Fig. 4C), though the OCCs underwent expansion [23].
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Incorporation of HCs in Porcine Cumulus Matrix Is Independent from Oocytes
Having established that OCCs are able to promote the incorporation of HCs in the matrix, we next investigated if the oocyte has a role in this process. For this, OOXs were cultured with FSH in the presence of pig serum. As shown in Figure 5, free heavy chains were detected by II antibody in the expanded matrix of the OOXs. These results indicate that the formation of HC-HA complexes does not require the presence of the oocyte.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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I family are fundamental for formation and stability of cumulus matrix and for female fertility [1, 7]. Previous studies suggest that transfer of these molecules from the blood to the follicular fluid is temporally and selectively regulated [9, 10]. A molecular sieve blood-follicle barrier in mammalian ovaries has been proposed for many years. In fact, comparative analysis of protein concentrations in serum and follicular fluid indicated that serum molecules with a size of less than 500 kDa entered the follicles, while those larger were excluded [28]. Although II-related molecules have a molecular weight ranging from 220 to 120 kDa, they were detected by immunoblotting in follicular fluid collected after, but not before, hCG injection in mice [9]. This observation led to the conclusion that LH/ hCG-induced changes in permeability of ovarian vasculature were essential for their diffusion into the follicular fluid. The unique exclusion of II-related molecules from the ovarian unstimulated follicles were imputed to net negative charges of II molecules [10]. However, here we show, by using the same technique and antibody used for II detection in mouse follicular fluids, that follicular fluids of medium-sized follicles of prepuberal gilts and those of large follicles of eCG-primed gilts contain high levels of II family members circulating in the serum. Therefore, it appears to be no barrier to the transfer of these molecules from the capillaries to the follicular antrum. In agreement with the reported increase of vasculature permeability and edema formation in the theca of periovulatory follicles in several species [28], an increase of II concentration was detected in pig follicular fluid at 24 h post-hCG injection. Thus, it might be that difficulty in retrieving substantial amounts of mouse follicular fluid hindered the detection of II before hCG but not after hCG. However, species-dependent differences between the mouse and pig blood-follicle barrier cannot be excluded. The presence of II molecules in the pig follicular fluid of medium-sized follicle would explain its ability to support full expansion of pig OCCs in vitro in the absence of serum [27].
HCs of II-related molecules are covalently linked to HA during in vivo mouse cumulus expansion and significantly contribute to cumulus matrix stability [1, 2]. Results reported in the current paper show that HC-HA covalent complexes are formed during in vivo porcine cumulus expansion as well. This is supported by the evidence that, besides native II-related molecules, single HCs are present in the cumulus matrix and that they cannot be dissociated from the matrix under reducing conditions without prior digestion of the samples with Streptomyces hyaluronidase.
Interestingly, although fetal bovine serum contains II molecules and is mainly used for in vitro OCC expansion studies, Western blot analysis with human II antibody failed to detect single HCs in hyaluronidase extracts of mouse OCCs expanded in vitro in this culture condition [11]. This observation questioned the ability of OCCs to autonomously promote the covalent transfer of HCs from II to HA and favored the hypothesis that a granulosa cell-derived factor(s) facilitate this reaction in the follicle. We obtained similar negative results by culturing pig OCCs in the presence of fetal bovine serum. However, HC-HA complexes could be detected in the matrix of in vitro-expanded OCCs when pig serum or pig follicular fluids were added to the medium as a source of II molecules. Reasons for this discrepancy might be due to species differences in HC antigenicity and inability of the commercially available antibody generated against human II to efficiently recognize bovine HCs. This hypothesis is also supported by the evidence that antibodies generated against bovine HCs could not reveal human HCs in Western blot [29]. In any event, positive results obtained with pig serum and follicular fluid definitely demonstrate that pig OCCs synthesize the factor mediating covalent transfer of HCs from II molecules to HA. This result is in agreement with high expression of TNFIP6 by cumulus cells in mouse and rat follicles after hCG [14, 17]. Indeed, TNFIP6 is a catalyzer of this reaction because it promotes the formation of HC-HA complexes when added to a mixture of HA and mouse serum, and disruption of TNFIP6 gene in mice prevents HC-HA complex formation in the preovulatory OCCs, leading to defects in cumulus matrix organization and stability [2]. The TNFIP6 gene is also expressed by granulosa cells in hCG-stimulated follicles, although at a lower level than in cumulus cells [14, 17], and medium conditioned by granulosa cells can promote HC transfer from II to HA in a cell-free system [11]. Therefore, granulosa cells could enhance the formation of HC-HA complexes in OCCs expanded in vivo. It has been reported that HC-HA coupling activity significantly increases in follicular fluid after hCG [12]. However, results reported here show that 10% follicular fluid collected from pig follicles at 24-h hCG was ineffective in increasing HC transfer in OCCs cultured in vitro.
It has been well established that mouse cumulus cells require oocyte-soluble factors together with hormone stimulus to synthesize structural components of the expanded matrix [3, 15], and oocyte-derived growth differentiation factor 9 has been implicated in this function as well as in the expression of TNFIP6 [17, 30]. In contrast, synthesis and retention of HA by pig cumulus cells normally occur in the absence of oocyte [20, 22, 23]. Accordingly, we show here that covalent linkage of HC to HA is promoted in OOXs with the same efficiency as in intact OCCs. This result confirms and extends previous findings demonstrating that pig cumulus cells are independent from oocyte both in HA synthesis and organization.
In conclusion, our studies provide evidence that HCs of II are covalently linked to HA in pig OCCs expanded in vivo, thereby directly participating to the formation and stability of cumulus matrix. In vitro studies indicate that pig cumulus cells produce the factors required for completing this reaction independently from the oocyte. We also show that II molecules can freely cross the blood-follicle barrier and that follicular fluid can be successfully used for stabilizing in vitro-expanded matrix.
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FOOTNOTES |
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2 Correspondence: Nagyova Eva, Academy of Sciences of the Czech Republic, Institute of Animal Physiology and Genetics, Rumburska 89, 277 21 Libechov, Czech Republic. FAX: +420 315 63 95 10; nagyova{at}iapg.cas.cz
Received: 15 March 2004.
First decision: 6 April 2004.
Accepted: 21 July 2004.
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