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Regular Article |
a Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTION MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cumulus cells, follicle, gene regulation, meiosis, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTION MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Whereas the preovulatory LH surge triggers the cumulus expansion of preovulatory follicles in vivo, cumulus cells are capable of undergoing expansion in response to FSH in vitro [2]. FSH increases levels of intracellular cAMP, activators of cAMP-dependent protein kinase, and epidermal growth factor (EGF), which activates the tyrosine kinase cascade [10–13]. Serum and follicular fluid contain factors that stimulate the synthesis of HA, a matrix constituent, and stabilize the organization of the cumulus extracellular matrix [14–18].
In vitro studies on interactions between the oocyte and the follicular cells have demonstrated an active role for the oocyte in the regulation of cumulus cell function. Mouse oocytectomized COCs (OXCs) show a diminished ability to synthesize HA or a failure of expansion in cumulus cells after stimulation with FSH or EGF as compared with intact COCs [19]. Incubation of mouse cumulus cells with isolated oocytes (coculture) or in denuded oocyte-conditioned medium stimulates FSH-induced HA synthesis and expansion [19, 20]. These reports demonstrated that the secretion of a soluble factor from oocytes is involved in HA synthesis and cumulus expansion in vitro and that this oocyte-secreted factor acts independently or downstream of the cAMP regulation. Porcine and bovine oocytes also secrete a cumulus expansion-enabling factor, although porcine cumulus expansion progresses steadily without this factor [21–23]. Mouse OXCs are stimulated to expand in culture medium conditioned by porcine cumulus and mural granulosa cells isolated from the preantral stage to the preovulatory stage, indicating that these somatic cells are capable of producing the cumulus expansion-enabling factor [24]. The factor secreted from porcine granulosa cells is not identical to that secreted from mouse oocytes, but these facts suggest that regulation of porcine cumulus expansion by both oocyte-secreted factor and cumulus- or mural granulosa cell-secreted factor is different from that in rodents.
Recently, three kinds of mammalian genes encoding putative plasma membrane HA synthases (HAS1, HAS2, and HAS3) have been identified [25–29]. Several mammalian cell lines transfected with their cDNAs exhibit a marked increase of HA production, indicating an important role for these enzymes in HA biosynthesis. Sequence comparison has indicated low levels (55%–71%) of amino acid identity among the three HA enzymes within species, in contrast to the high degree (96%–99%) of conservation between human and mouse [29]. Potential differences between these synthases in function have been little studied. Furthermore, the factors modulating their expression have hardly been investigated.
The cell surface glycoprotein CD44, the principal HA receptor, is present on mature oocytes and preimplantation embryos in mammals [30, 31]. The addition of HA or chondroitin sulfate A, a ligand of CD44, to culture medium improves the development of in vitro-matured/fertilized porcine embryo to the blastocyst stage [32]. These facts suggest that CD44 interacts with these glycosaminoglycans (GAGs) and has a positive role in oogenesis and embryogenesis.
We herein describe the mRNA expression of two kinds of HA synthases and of CD44 in porcine COCs, OXCs, and oocytes, and the localization of CD44 protein in COCs during in vitro maturation. The present study was conducted to examine 1) which of the HA synthase genes are involved in cumulus expansion, 2) the effect of eCG and porcine follicular fluid (pFF) on the expression of HA-related genes and the CD44 gene, 3) the effect of oocyte-secreted factor on the transcription of these genes, and 4) the localization of CD44 in COCs. The long-term objective of our ongoing study is to investigate the role of the HA-CD44 system as it relates to porcine oocyte maturation or oocyte viability.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTION MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals and supplies were obtained from the following sources: Ca- and Mg-free Dulbecco PBS from Nissui Pharmaceutical Co. (Tokyo, Japan); medium 199 and the oligonucleotide primers from Gibco BRL Life Technologies Inc. (Grand Island, NY); BSA, sodium pyruvate, mineral oil, polyvinyl-pyrrolidone (PVP), and hyaluronidase from Sigma Chemical Co. (St. Louis, MO); penicillin G potassium and streptomycin sulfate from Meiji Seika Co. (Tokyo, Japan); eCG from Teikoku Zouki Pharmaceutical Co. (Tokyo, Japan); RNeasy mini-kit to isolate total RNA from QIAGEN K.K. (Tokyo, Japan); deoxyribonuclease I from Stratagene Co. (La Jolla, CA); Ready-To-Go reverse transcription-polymerase chain reaction (RT-PCR) beads from Amersham Pharmacia Biotech Inc. (Piscataway, NJ); BigDye Terminator Cycle Sequencing FS Ready Reaction Kit from Perkin Elmer Applied Systems (Foster City, CA); CD44 monoclonal antibody (i.e., anti-porcine CD44 mouse immunoglobulin G [IgG]) (cell line, PORC24A) from VMRD Inc. (Pullman, WA); fluorescein isothiocyanate (FITC)-conjugated goat affinity purified F(ab') fragment to mouse IgG as the secondary antibody from ICN Pharmaceuticals Inc. (Aurora, OH); and all other reagents from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). PBS containing 3 mg/ml BSA, 100 IU/ml penicillin, and 100 µg/ml streptomycin (PBS-BSA) was prepared for washing COCs, OXCs, and oocytes and for dilution of antibodies for immunofluorescence. PBS containing 3 mg/ml PVP and antibiotics (PBS-PVP) was prepared for fixing samples. The arrangement of pFF was as follows. Collected ovaries from prepubertal gilts were transported at 37°C from a local abattoir to the laboratory within 1 h. Follicular contents were aspirated from antral (2–5 mm in diameter) follicles and centrifuged at 1500 x g at 10°C for 20 min. The supernatant was stored at -20°C.
In Vitro Maturation and Oocytectomy of Porcine COCs
Follicular contents were obtained from antral (2–5 mm in diameter) follicles by aspiration, and then only oocytes with compact, multilayered cumulus cells and a uniformly granulated cytoplasm were collected as cultivable COCs. After a brief wash in PBS-BSA, COCs were placed in each culture medium. The following four types of culturing using medium 199 were carried out: 1) medium 199 alone, 2) medium supplemented with 10 IU/ml eCG, 3) medium supplemented with 10% (v/v) pFF, and 4) medium supplemented with both 10 IU/ml eCG and 10% (v/v) pFF. Medium 199 with Earle salt and L-glutamine contained 2.2 mg/ml sodium bicarbonate, 0.1 mg/ml sodium pyruvate, 10 mg/ml BSA, 100 IU/ml penicillin, and 100 µg/ml streptomycin.
For oocytectomy, a COC was fixed to the bottom of the dish with precision tweezers, and a prick was made through the layer of cumulus cells and the oocyte with the tip of a 26-gauge needle. Then the oocytoplasm was pressed out gradually with the tweezers. Almost all of the oocyte contents were readily removed in this way. OXCs with essentially the shape of intact COCs without oocytoplasm were cultured in the same medium as COCs. The COCs and OXCs were incubated at 37°C for 48 h in a humid atmosphere of 5% CO2 in air. Each droplet of medium (200 µl) was overlaid with mineral oil and contained approximately 20 COCs or OXCs. In some COCs, cumulus cells were removed with 0.1% (w/v) hyaluronidase, and the oocytes were washed three times with PBS-BSA.
Morphologic Assessment of Cumulus Expansion
Fifty-five COCs or OXCs in each treatment were evaluated for the degree of cumulus expansion at 0, 12, 24, 36, and 48 h after culture. Assessment of cumulus expansion was based on the size of the area occupied by the cumulus cell aggregate of each COC or OXC. The length and width of a COC or OXC were measured with an eyepiece micrometer under an inverted microscope equipped with Nomarski optics (100x). The length was defined as the distance between the two most broadly separated points, and the width was defined as the distance between the two closest points. The area occupied by a COC or OXC was calculated using the following formula: area = length x width x 0.7854 [18]. Experiments were carried out three times, and the results were presented as the mean ± SEM area (expressed in units of 10-3 mm2) for each treatment.
Isolation of Total RNA From COCs, OXCs, and Oocytes
Fresh COCs or OXCs, and COCs or OXCs cultured in each medium for 24 h, COCs or OXCs cultured in the medium supplemented with both eCG and pFF for 48 h, and cumulus cell-free oocytes after a 24-h culture with both eCG and pFF were collected for extraction of total RNA. Total RNAs were isolated according to the instructions supplied with the RNeasy mini-kit. Briefly, samples were lysed and homogenized in the presence of a highly denaturing guanidinium isothiocyanate-containing buffer, then adjusted with ethanol. This solution was applied to a spin column in which the total RNA binds to the membrane by centrifugation. The total RNA was treated with 83 U of DNase I on the spin column at room temperature for 15 min and eluted in diethyl pyrocarbonate-treated distilled water after the washing away of contaminants. With this procedure, 0.3–2 µg of total RNA was obtained from 250 COCs or OXCs.
Semiquantitative Reverse Transcription-Polymerase Chain Reaction
Reverse transcription-polymerase chain reaction (RT-PCR) was performed using Ready-To-Go RT-PCR beads that were optimized to allow the first-strand cDNA synthesis and PCR reactions to proceed sequentially as a single-step reaction and using a PCR Thermal Cycler TP2000 (TaKaRa Co., Kyoto, Japan). Each primer was designed according to published mouse cDNAs, including regions highly conserved between the mouse and humans. For has2, the sense primer (5'-GAATTACCCAGTCCTGGCTT-3') and the antisense primer (5'-GGATAAACTGGTAGCCAACA-3') generated a 581-base pair (bp) cDNA fragment correspondent to mouse Has2 nucleotides (accession number: U52524). For has3, the sense primer (5'-CCTACTTTGGCTGTGTGCAA-3') and the antisense primer (5'-AGGCTGGACATATAGAGAAG-3') generated a 525-bp cDNA fragment correspondent to mouse Has3 nucleotides (accession number: U86408). For CD44, the sense primer (5'-GTACATCAGTCACAGACCTAC-3') and the antisense primer (5'-CACCATTTCCTTGAGACTTGCT-3') generated a 598-bp cDNA fragment correspondent to mouse CD44 nucleotides (accession number: M27129). For ß-actin (as an internal positive control), the sense primer (5'-GACCCAGATCATGTTTGAGACC-3') and the antisense primer (5'-ATCTCCTTCTGCATCCTGTCAG-3') generated a 593-bp cDNA fragment correspondent to mouse ß-actin nucleotides (accession number: X03672). Fifty nanograms of total RNA extracted from COC or OXC mRNA, the equivalent of total RNA from 30 oocytes, was reverse transcribed and then PCR amplified, in a total reaction volume of 50 µl containing 10 pmol of each sense and antisense primer, 0.5 µg of oligo(dT)12–18 primer, 
2.0 U of Taq DNA polymerase, 10 mM Tris-HCl (pH 9.0), 60 mM KCl, 1.5 mM MgCl2, 200 µM of each dNTP, Moloney murine leukemia virus reverse transcriptase (FPLCpure), and RNAguard ribonuclease inhibitor (porcine) and stabilizer, including ribonuclease/deoxyribonuclease-free BSA. This mixture, overlaid with mineral oil, was incubated at 42°C for 20 min for the RT reaction. PCR amplification proceeded after inactivation of the reverse transcriptase by heating at 95°C for 5 min. PCR cycling conditions were as follows: COCs and OXCs—ß-actin and CD44: 25 cycles of 1-min denaturation at 95°C, 1-min annealing at 55°C, 1-min extension at 72°C; has2: 26 cycles of 1-min denaturation at 95°C, 1-min annealing at 55°C, 1-min extension at 72°C; has3: 28 cycles of 1-min denaturation at 95°C, 1-min annealing at 55°C, 1-min extension at 72°C; oocytes—30 cycles of 1-min denaturation at 95°C, 1-min annealing at 58°C, 1-min, 30 sec-extension at 72°C. For semiquantitative PCR, their cycle numbers were optimized to ensure amplification of cDNA in the exponential phase of PCR. To detect genomic DNA contamination, total RNA was subjected to RT-PCR without reverse transcriptase using ß-actin primer pairs. The amplified product was electrophoresed on 2% agarose gel and visualized by ethidium bromide staining. The intensity of the objective bands was quantified by densitometric scanning using NIH Image version 1.62 free software (NIH, Bethesda, MD). The respective values of has2, has3, and CD44 were normalized according to those of ß-actin to evaluate arbitrary units of the relative abundance of the targets.
DNA Sequencing
Direct sequencing was carried out to identify RT-PCR products using a fluorescent dye terminator and the ABI PRISM 310 Genetic Analyzer (Perkin-Elmer Corporation). According to the instructions supplied with the sequencing kit, the cDNA fragments were mixed with four dye-labeled dideoxynucleotides as terminators, a thermally stable AmpliTaq DNA polymerase, and sense or antisense primer, then set on the thermal cycler for the sequencing reaction. Extended products were analyzed on the DNA sequencer.
CD44 Localization by Immunofluorescence
Monoclonal antibody to CD44 was used for the investigation of CD44 localization. COCs cultured in each treatment for 24 h were fixed in 2% (v/v) formaldehyde in PBS-PVP at 4°C for 30 min and then washed three times with PBS-BSA. The specimens were permeabilized in 0.25% (v/v) Tween 20 in PBS-BSA at room temperature for 2 min, washed three times in PBS-BSA, and then stored in PBS-BSA containing 0.02% sodium azide at 4°C for 0–7 days. Fixed samples were incubated with primary antibody diluted 1:50 in PBS-BSA at 37°C for 2 h. After several washes with PBS-BSA containing 0.5% Triton X-100 (PBS-BSA-T), these samples were exposed to FITC-conjugated goat anti-mouse antibody diluted 1:200 in PBS-BSA at 37°C for 1 h. To detect nuclear localization, propidium iodide (PI) staining was followed by several washes with PBS-BSA-T. Stained samples were incubated in 10 µg/ml PI at 37°C for 1 h, washed with PBS-BSA-T, and then mounted under a coverslip with PBS-BSA. Whole mounted specimens were placed on an Axioplan Zeiss microscope (Carl Zeiss Co., Oberkochen, Germany) and examined using a Biorad MRC-1024 laser scanning confocal microscope (Bio-Rad Laboratories Co., Hercules, CA).
Statistical Analysis
The area of cumulus expansion was presented as the mean ± SEM (10-3 mm2) for each treatment within the indicated time periods. The effects of eCG and pFF on cumulus expansion were analyzed by one-way factorial ANOVA among four groups of COCs or OXCs at the same time point, and multiple comparisons were made with the Bonferroni t-test. The effects of oocytectomy on cumulus expansion was also analyzed by the Student t-test between COCs and OXCs at the same time point in the same treatment. The densitometry ratio in the expression of has2, has3, and CD44 mRNAs was analyzed by one-way factorial ANOVA between the fresh and expanded COCs and OXCs (culture period 0 h, 24 h with or without eCG and pFF), followed by the Bonferroni t-test. P values less than 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTION MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this experiment, we assessed the effects of eCG, pFF, and oocytectomy on cumulus expansion in porcine COCs and OXCs. Photographs of COCs that illustrate the actual nature of a typical cumulus expansion in vitro are presented in Figure 1. The areas occupied by COCs or OXCs in each group were measured every 12 h for a 48-h culture (Fig. 2). Cumulus expansion was already observed at 12 h of culture in the medium containing eCG and pFF. The group supplemented with both eCG and pFF showed the most prominent cumulus expansion, followed by that supplemented with pFF alone and that supplemented with eCG alone. These findings indicate a synergistic effect of eCG and pFF. Of particular note, the area had increased markedly at 48 h after culture in the medium with eCG and pFF (COCs: 255.4 ± 30.1 x 10-3 mm2; OXCs: 201.9 ± 20.4 x 10-3 mm2) compared with culture in the medium alone (COCs: 29.8 ± 1.2 x 10-3 mm2; OXCs: 31.2 ± 1.0 x 10-3 mm2). The addition of pFF alone caused the outer layer of the complexes to scatter after 24 h of culture. On the other hand, the addition of eCG alone induced expansion but did not cause the dispersion of the outer layer as in the groups treated with pFF alone and both eCG and pFF. Although no differences were observed between COCs and OXCs cultured in the medium alone or supplemented with pFF, the effect of oocytectomy was significantly different in the medium supplemented with eCG alone, suggesting that the oocyte influences eCG-induced expansion of cumulus cells but may not participate in pFF-induced expansion. When COCs and OXCs were cultured in medium alone, they showed no evidence of cumulus expansion during the culture period, and cumulus cells gradually disassociated from oocytes after 36 h of culture.
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Expressions of HA Synthase (has2 and has3) and CD44 mRNAs in COCs, OXCs, and Oocytes
To investigate whether expression of the two HA synthase genes is involved in cumulus expansion and whether the expression of the CD44 gene is related to expression of has2 and has3, we confirmed the expression of mRNA in COCs, OXCs, and oocytes using RT-PCR. In COCs, although the patterns of individual expression were diverse under each culture condition, all transcripts were detectable with amplified products of near predicted sizes, while the transcription of has3 did not occur in OXCs (Fig. 3). We also confirmed the expression of these genes in cumulus cells and oocytes cultured with eCG and pFF for 24 h (Fig. 4). These findings demonstrate that has2 and CD44 mRNAs are expressed in cumulus cells (oocytectomized COCs), whereas has3 mRNA is expressed only in oocytes. The transcripts of CD44 were detectable in fresh (0 h) COCs; has2 mRNA, however, was not really detectable until 24 and 48 h after culture (Fig. 3). The levels of has2 and CD44 mRNAs increased significantly after 24 h of culture in the presence of eCG or pFF compared with medium alone (without the expression of CD44 in OXCs cultured with eCG), indicating that these genes have a positive dependency on eCG and pFF. In particular, the has2 mRNA was induced more strongly by eCG than by pFF. CD44 mRNA tended to be expressed more dominantly in the presence of both eCG and pFF compared with eCG alone or pFF alone. In contrast, a high level of expression of has3 mRNA was found in COCs in medium alone. In medium supplemented with eCG and pFF, expression of the has3 transcript decreased with cumulus expansion. Oocytectomy slightly reduced the expression of has2 mRNA in a 24-h culture with eCG and pFF. On the other hand, no significant difference was seen between COCs and OXCs in the expression of CD44 mRNA. The expression of has2 mRNA decreased at 48 h of culture in COCs (compared with 24 h of culture) in the presence of both eCG and pFF. However, its expression increased in OXCs.
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DNA Sequencing
Partial cDNAs were amplified by RT-PCR from porcine COCs, OXCs, and oocytes, using primers complementary to the mouse Has2, Has3, and CD44 sequences. The DNA products were identified by direct sequencing with the dye terminator method. Each sequence was compared for homology with the mouse and human cDNA sequences. The similarity of the partial porcine has2 sequence was 74.9% to mouse Has2 and 80.4% to human HAS2 (Fig. 5). The porcine has3 fragment showed a high degree of similarity to mouse Has3 and human HAS3 with over 90% identity (Fig. 6). The homology of the porcine CD44 cDNA amplified was 77% with mouse and 79% with human CD44 (Fig. 7). Sequence comparisons indicate that the partial porcine cDNAs amplified with mouse Has2, Has3, and CD44 primers conformed to porcine has2, has3, and CD44, respectively.
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CD44 Localization by Immunofluorescence
The localization of CD44 in COCs was determined by indirect immunofluorescence. The specificity of the immunoreactivity was demonstrated by staining with only FITC-conjugated goat anti-mouse antibody (secondary antibody) or incubation in PBS-BSA (data not shown). There was no apparent staining in fresh COCs, but strong signals were observed in COCs cultured with eCG and pFF for 24 h (Fig. 8). The positive staining was found to be present on the cytoplasm along the perimembrane in cumulus cells and at the junctions between cumulus cells and oocytes (Figs. 9 and 10). Staining of heterogeneous patches, such as the foot of junctions between cumulus cells and oocytes, was also found on the surface of oocytes or inside of zona. We also observed heterogeneous cytoplasmic staining in some oocytes (Fig. 8). The cytoplasmic staining of oocytes seemed to be inclined to find in immature (0 h) COCs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTION MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-trypsin inhibitors that stabilize the cumulus extracellular matrix [15, 34] and many elements [35]. The conspicuous expansion of COCs in the presence of both eCG and pFF suggests not only a synergistic effect of stimulation of HA synthesis by eCG and pFF, but also a stabilization of HA by pFF. The effect of oocytectomy was consistently found in only the eCG treatment group when comparing cumulus expansion between COCs and OXCs (Fig. 2), suggesting that the porcine oocyte is involved in eCG-induced expansion of cumulus cells. We did not see notable effects of oocytectomy in the pFF treatment group and the combined eCG and pFF treatment group (except for 36 h of combined eCG and pFF treatment). Cumulus expansion depends on some factor secreted by the oocyte mediating FSH, cAMP, or EGF in mice [19, 20]. Our findings support previous reports [21–23] that demonstrated the presence of an oocyte-derived factor that stimulates cumulus expansion in the presence of FSH or EGF as seen in mice; however, this factor is not essential for the expansion of porcine and bovine cumulus cells. In this study, we investigated the expressions of the HA synthase family and CD44 mRNAs in COCs, OXCs, and oocytes during cumulus expansion stimulated in vitro. The results demonstrated that HA synthase mRNAs are expressed not only in cumulus cells (has2) but also in oocytes (has3), suggesting that oocytes, in addition to cumulus cells, synthesize HA (Fig. 4).
The expression of has2 mRNA was induced by eCG and pFF, which brought about cumulus expansion (Fig. 3). Fulop et al. [36] reported that mouse Has2 is expressed during COC matrix expansion induced by hCG injection and that the pattern of increased Has2 mRNA levels is similar to that observed in HA synthesis in vivo. This evidence of Has2 transcription suggests a role for HAS2 in HA synthesis accompanied by cumulus expansion. In our experiments, the level of has2 mRNA in COCs and OXCs did not always conform to the degree of cumulus expansion simultaneously. This evidence suggests that the cumulus expansion consists of both the synthesis of HA and other matrix components and their deposition around the cumulus cells.
In COCs after 24 h of culture, the expression of has3 mRNA remarkably increased in the medium-alone group compared with the eCG-alone, pFF-alone, and combined eCG and pFF groups. The pattern of has3 mRNA expression was different from that of has2 mRNA (Fig. 3). We also confirmed has3 mRNA expression in oocytes removed from cumulus cells at 0, 24, and 48 h after culture in oocytes incubated in the medium alone or in medium supplemented with eCG and pFF (data not shown). We did not find any apparent difference of has3 transcription level between conditions or culture periods. Therefore, it seems that the regulation of has3 mRNA expression is distinct from that of has2 mRNA expression.
It is not clear what part of the has3-derived HA the oocyte takes in. Several studies have demonstrated that the addition of HA to culture media effectively prevents fragmentation or segmentation of oocytes and supports the development of in vitro-matured, -fertilized, and -cultured oocytes to the blastocyst stage [32, 37]. Sato et al. [38] showed that specific GAGs purified from pFF, the fraction with a retention time nearly coincident with that of HA, promotes viability of oocytes. Hess et al. [3] demonstrated that disruption of cumulus expansion by excess HA affects the early development of embryos derived from COCs stored within the oviduct. These results indicate that HA positively participates in the development of oocytes and embryos, and thus, it is tempting to speculate that oocytes themselves produce HA to maintain development and viability [9].
Spicer et al. [29] suggested that potential differences in the function of each HAS protein (HAS1, HAS2, and HAS3) could be related to the length of the HA chain synthesized, the rate of HA synthesis, the ability to interact with cell type-specific accessory proteins, and whether or not the HA is preferentially secreted by the cell or alternatively retained by the cell in the form of a pericellular coat. Recently, enzymatic properties of three HAS proteins have been reported by Itano et al. [39]. Kinetic studies of these enzymes in the membrane fraction isolated from HAS transfectants (the host cells were COS-1 cells or rat 3Y1 fibroblasts) demonstrated that each HAS protein is distinct in enzyme stability, elongation rate of HA, and apparent K(m) values for two substrates, i.e., UDP-GlcNAc and UDP-GlcUA. When HAs generated by recombinant HAS proteins in vitro were compared, it was found that the molecular mass of HA synthesized by HAS3 (1 x 105 to 1 x 106 Da) was smaller than that of HA synthesized by HAS1 and HAS2 (2 x 105 to 2 x 106 Da). The expressions of each HAS protein are restricted based on the developmental stage of embryos in the mouse and Xenopus laevis, and they differ among human adult tissues [40]. This would suggest distinctive roles and functions for each HA generated by respective HAS genes. Further functional evaluation of HAS and HAS-derived HA is needed to elucidate the roles of HA molecules in signal transduction with linkage to receptors or proteoglycans and in the architecture of the extracellular matrix.
Oocytectomy decreased the level of has2 mRNA, indicating that the porcine oocyte up-regulates has2 mRNA expression (Fig. 3). This finding was consistent with the results of previous studies in which mouse oocytes stimulated HA synthesis downstream from the step involving cAMP generation induced by hormones [19, 20]. However, the oocyte was not essential for has2 mRNA expression in porcine cumulus cells. Nakayama et al. [41] demonstrated that oocytectomy reduces HA synthesis but does not stop cumulus expansion in porcine COCs because chondroitin sulfate (CS) is synthesized independently. They speculated that HA synthesis is dependent on the oocytes, whereas CS synthesis is a basic response of cumulus cells. Our findings suggest that the expression of has2 mRNA during cumulus expansion is induced directly by gonadotropins and via an oocyte-derived factor. The properties of this oocyte factor are mimicked by transforming growth factor ß1 (TGFß1), which stimulates HA synthesis to nearly the same level as that in oocyte-conditioned medium. However, anti-TGFß1 does not inhibit the response to oocyte factor- or FSH-induced HA synthesis in COCs [4]. These results suggest that the oocyte factor is not a kind of the TGFß1 but rather is related to the TGFß family. Recently, Elvin et al. [42] reported that recombinant growth differentiation factor 9 (GDF-9), a member of the TGF superfamily that shows a high level of expression in mammalian oocytes, induces cumulus expansion in vitro. They found that recombinant GDF-9 stimulates the expression of HAS2, cyclooxygenase 2, and steroidogenic acute regulator protein mRNA, but suppresses the expression of urokinase plasminogen activator and LH receptor mRNA. These findings indicate that GDF-9 acts as an oocyte-secreted paracrine factor to modulate downstream target genes involved in cumulus expansion and maintains the oocyte microenvironment for normal ovulation, fertilization, and female reproduction.
The expression of CD44 mRNA was detected by RT-PCR in cumulus cells (Fig. 4) but not in oocytes. Equine chorionic gonadotropin or pFF stimulated the expression of CD44 mRNA to the same level in COCs, and combined stimulation with eCG and pFF further enhanced expression (Fig. 3). The presence of oocytes did not affect CD44 mRNA expression in the pFF-alone or combined eCG and pFF treatment groups, but the presence of oocytes tended to increase expression in the eCG-alone treatment group. The pattern of CD44 mRNA expression seemed to be related to the expansion area after 24 h of culture. It may be supposed that the expression of CD44 is stimulated not only by HA but also by the ligand for CD44 and other GAGs that affect the HA-binding ability of CD44 [43].
Campbell et al. [31] demonstrated by indirect immunofluorescence that CD44 is present on the surface of human oocytes and cumulus cells and continues to be expressed on the embryonic surface until the prehatched blastocyst stage. We detected immunoreactivity of CD44 on the surface or in the cytoplasm of oocytes in addition to cumulus cells (Figs. 8–10); however, CD44 mRNA was not found on oocytes. A possible explanation for this contradiction is that we did not verify the expression of the alternative spliced exons that are involved in the functional heterogeneity of CD44 using RT-PCR. Genomic analysis of CD44 showed the existence of 20 exons, which by alternative RNA splicing code for the CD44 standard/hemopoietic form (CD44s/CD44H) and numerous variant isoforms (CD44v) [44, 45]. CD44s is composed of the following exons: exons 1s to 7s, encoding the extracellular domain; exon 8s (the transmembrane region); and exons 9s and 10s, which can be alternatively spliced, resulting in generation of a cytoplasmic tail [46]. The variant exons 1v to 10v located between exons 5s and 6s give enormous variability to the CD44 family of molecules. In the present study, the part of the CD44 cDNA that was amplified was restricted to exons 3s to 10s. It has been reported that the CD44 expressed in cumulus cells is mainly the standard form (CD44s) [47]. The positive reactivity to CD44 antibody in oocytes may indicate the expression of CD44 variants.
No studies have established the role of the HA-CD44 system in oocyte maturation. Recently, it has been shown that the degradation product of HA (3–10 disaccharides) induces the phosphorylation of the CD44 receptor, leading to the activation of a cytoplasmic kinase cascade, i.e., Raf-1 kinase, mitogen-activated protein kinase (MAPK) kinase (MEK-1), and extracellular signal-regulated kinase (ERK-1), which is subsequently translocated to nucleus [48]. Ras recruits Raf to the complex, resulting in Raf activation. Raf then activates the MAPK cascade by phosphorylation of MEK and MAPK [49]. This cascade is important for mitogenic signal transduction and sufficient for the induction of cell proliferation through the stimulation of protooncogenic transcription factors (such as ATF-2, c-Fos, c-Jun, c-Myc, ELK-1, and NF-I16) [50]. The MAPK cascade also plays a crucial role in activation and stabilization of the M-phase promoting factor during oocyte maturation [51–53].
In conclusion, we have provided evidence of different expression patterns between has2 and has3 mRNAs in porcine COCs during in vitro maturation. Our results suggest that the expression of has2 mRNA in cumulus cells is stimulated by eCG and pFF and that oocytes promote its expression. HAS2 is involved in HA synthesis during cumulus expansion. On the other hand, has3 mRNA is expressed in oocytes. The expression of CD44 mRNA is also stimulated by eCG and pFF. Immunoreactivity to CD44 is observed on the cytoplasm along the perimembrane of cumulus cells, at the junctions between oocytes and cumulus cells, and on the surface or in the cytoplasm of oocytes. Further studies are necessary to characterize the role of HA-CD44 signaling in oocyte maturation during cumulus expansion and in the maintenance of oocyte viability.
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FOOTNOTES |
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1 Supported by the Japan Society for the Promotion of Science (JSPS) grant RFTF 97L00904 of the "Research for the Future" Program and of the Program for Promotion of Basic Research Activities for Innovative Bioscience. 
2 Correspondence: Naoko Kimura, Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. FAX: 81 22 717 8879; naonao{at}bios.tohoku.ac.jp
Accepted: November 6, 2001.
Received: May 8, 2001.
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 M. Shimada, Y. Yanai, T. Okazaki, N. Noma, I. Kawashima, T. Mori, and J. S. Richards Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization Development, June 1, 2008; 135(11): 2001 - 2011. [Abstract] [Full Text] [PDF]
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 N. Borg and M. Holland The effect of glycosaminoglycans on rat gametes in vitro and the associated signal pathway Reproduction, March 1, 2008; 135(3): 311 - 319. [Abstract] [Full Text] [PDF]
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L. Nemcova, E. Nagyova, M. Petlach, M. Tomanek, and R. Prochazka Molecular Mechanisms of Insulin-Like Growth Factor 1 Promoted Synthesis and Retention of Hyaluronic Acid in Porcine Oocyte-Cumulus Complexes Biol Reprod, June 1, 2007; 76(6): 1016 - 1024. [Abstract] [Full Text] [PDF]
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D. W Erikson, A. L Way, D. A Chapman, and G. J Killian Detection of osteopontin on Holstein bull spermatozoa, in cauda epididymal fluid and testis homogenates, and its potential role in bovine fertilization Reproduction, May 1, 2007; 133(5): 909 - 917. [Abstract] [Full Text] [PDF]
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 K. Sayasith, K. A Brown, J. G Lussier, M. Dore, and J. Sirois Characterization of bovine early growth response factor-1 and its gonadotropin-dependent regulation in ovarian follicles prior to ovulation. J. Mol. Endocrinol., October 1, 2006; 37(2): 239 - 250. [Abstract] [Full Text] [PDF]
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H. G Clarke, S. A Hope, S. Byers, and R. J Rodgers Formation of ovarian follicular fluid may be due to the osmotic potential of large glycosaminoglycans and proteoglycans Reproduction, July 1, 2006; 132(1): 119 - 131. [Abstract] [Full Text] [PDF]
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Y. Miyake, H. Matsumoto, M. Yokoo, K. Miyazawa, N. Kimura, W. A. S. Tunjung, T. Shimizu, H. Sasada, H. Aso, T. Yamaguchi, et al. Expression and Glycosylation with Polylactosamine of CD44 Antigen on Macrophages During Follicular Atresia in Pig Ovaries Biol Reprod, March 1, 2006; 74(3): 501 - 510. [Abstract] [Full Text] [PDF]
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E. Nagyova, A. Camaioni, R. Prochazka, and A. Salustri Covalent Transfer of Heavy Chains of Inter-{alpha}-Trypsin Inhibitor Family Proteins to Hyaluronan in In Vivo and In Vitro Expanded Porcine Oocyte-Cumulus Complexes Biol Reprod, December 1, 2004; 71(6): 1838 - 1843. [Abstract] [Full Text] [PDF]
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 M. Shimada, M. Nishibori, Y. Yamashita, J. Ito, T. Mori, and J. S. Richards Down-Regulated Expression of A Disintegrin and Metalloproteinase with Thrombospondin-Like Repeats-1 by Progesterone Receptor Antagonist Is Associated with Impaired Expansion of Porcine Cumulus-Oocyte Complexes Endocrinology, October 1, 2004; 145(10): 4603 - 4614. [Abstract] [Full Text] [PDF]
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M. Jo, L. E. Thomas, S. E. Wheeler, and T. E. Curry Jr Membrane Type 1-Matrix Metalloproteinase (MMP)-Associated MMP-2 Activation Increases in the Rat Ovary in Response to an Ovulatory Dose of Human Chorionic Gonadotropin Biol Reprod, April 1, 2004; 70(4): 1024 - 1032. [Abstract] [Full Text] [PDF]
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R. Marchal, M. Caillaud, A. Martoriati, N. Gerard, P. Mermillod, and G. Goudet Effect of Growth Hormone (GH) on In Vitro Nuclear and Cytoplasmic Oocyte Maturation, Cumulus Expansion, Hyaluronan Synthases, and Connexins 32 and 43 Expression, and GH Receptor Messenger RNA Expression in Equine and Porcine Species Biol Reprod, September 1, 2003; 69(3): 1013 - 1022. [Abstract] [Full Text] [PDF]
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M. Schoenfelder and R. Einspanier Expression of Hyaluronan Synthases and Corresponding Hyaluronan Receptors Is Differentially Regulated During Oocyte Maturation in Cattle Biol Reprod, July 1, 2003; 69(1): 269 - 277. [Abstract] [Full Text] [PDF]
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M. Stojkovic, O. Krebs, S. Kolle, K. Prelle, V. Assmann, V. Zakhartchenko, F. Sinowatz, and E. Wolf Developmental Regulation of Hyaluronan-Binding Protein (RHAMM/IHABP) Expression in Early Bovine Embryos Biol Reprod, January 1, 2003; 68(1): 60 - 66. [Abstract] [Full Text] [PDF]
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A. E. Stock, N. Bouchard, K. Brown, A. P. Spicer, C. B. Underhill, M. Dore, and J. Sirois Induction of Hyaluronan Synthase 2 by Human Chorionic Gonadotropin in Mural Granulosa Cells of Equine Preovulatory Follicles Endocrinology, November 1, 2002; 143(11): 4375 - 4384. [Abstract] [Full Text] [PDF]
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M. Yokoo, Y. Miyahayashi, T. Naganuma, N. Kimura, H. Sasada, and E. Sato Identification of Hyaluronic Acid-Binding Proteins and Their Expressions in Porcine Cumulus-Oocyte Complexes During In Vitro Maturation Biol Reprod, October 1, 2002; 67(4): 1165 - 1171. [Abstract] [Full Text] [PDF]
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