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Articles |
-Inhibitor Binding to Hyaluronan in the Cumulus Extracellular Matrix Is Required for Optimal Ovulation and Development of Mouse Oocytes1
a Molecular and Developmental Biology,
b Children's Hospital Research Foundation,
c Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inhibitor (II) family of serum glycoproteins and HA is necessary for optimal cumulus extracellular matrix (cECM) stabilization and cumulus expansion. Intravenous administration of HA oligosaccharides inhibited the binding of II to endogenous HA, disrupting the process of expansion and resulting in a reduction in the size of the cumulus mass. Western blot and immunocytochemical analyses of COCs from HA-treated animals demonstrated a reduction of II heavy chains within the cECM. Additionally, HA-treated immature animals ovulated 56.3% fewer COCs compared to control animals. The developmental potential of COCs in HA-treated animals was also tested. Extended periods of oviductal storage of COCs ovulated by HA-injected adult mice resulted in a reduction of normal embryos and a significant increase in the proportion of fragmented oocytes/embryos. These observations support the view that covalent binding of II heavy chains to HA is required for optimal cumulus expansion, extrusion of the COCs from the follicle at ovulation, and maintenance of oocyte viability within the oviduct.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The expanded cECM is stabilized through the binding of hyaluronan (HA) by serum glycoproteins of the inter--inhibitor family (II; [19–21]). Indeed, the compact preovulatory cumulus oocyte complex (COC) can form an organized and stabilized cumulus mass in vitro in the presence of serum, FSH, and substrates for HA synthesis. In the absence of serum, either purified II or pre--inhibitor (PI) is able to organize and stabilize the expanding cECM in vitro. II and PI possess a high net negative charge and are found within human blood at relatively high concentrations (0.5 mg/ml). Both proteins are composed of heavy and light chains linked via a chondroitin sulfate moiety. PI is composed of a single heavy chain and a light chain while II is composed of two heavy chains and one light chain [22]. While these proteins have been shown to play a critical role in cECM stabilization, II and PI do not enter the follicle until it responds to an ovulatory stimulus. The LH surge increases the permeability of the charge and size-selective blood follicle barrier, allowing these serum glycoproteins to enter the antral cavities of responding follicles [23, 24]. Once within the antral cavity, a factor(s) produced and/or secreted by the membrana granulosa catalyzes a conversion from a charge-mediated interaction to a covalent linkage between HA and II/PI heavy chains during cECM stabilization [21].
The physiological significance of an optimally expanded cumulus has been explored by disrupting the process of HA synthesis or its specific binding to II. Addition of exogenous HA (intact or oligosaccharides) to in vitro culture medium disrupts cumulus expansion by competitively inhibiting binding between endogenous HA and II or PI [20, 25]. In contrast, however, chondroitin sulfate, a glycosaminoglycan related to HA, has no effect on in vitro cumulus mass stability [20, 25]. Administration of 6-diazo-5-oxo-1-norleucine (DON), an inhibitor of glucosamine (a substrate for production of HA) synthesis, to hormonally primed animals blocks both cumulus expansion and ovulation, but it does not prevent rupture of the follicle wall [14, 26]. Consistent with these findings, animals treated with an interleukin-1 receptor antagonist apparently block ovulation through a similar mechanism [27]. These experiments taken together provide convincing support for the idea that HA synthesis and cumulus expansion are required for ovulation [26].
Related studies have shown that follicular rupture and ovulation are not explosive events but processes that occur under the influence of constant and steady intrafollicular pressure [28–32]. In hamsters, the entire ovulation and COC extrusion process has been found to occur over 10 min and is divided into two distinct phases. In the first, rapid phase (< 1 min), only ~25% of the COC volume passes through a protease-created rupture site located at the apex of the follicular stigma [31]. Thus, the COC acts to "plug" or seal the rupture site to maintain the constant intrafollicular pressure needed for extrusion. During the second, slow phase (up to 10 min), the constant pressure and smooth muscle contraction help to extrude the remaining ~75% of the fully expanded COC [30, 31]. Additionally, the viscoelastic properties of the cumulus mass apparently aid in protecting the oocyte from the mechanical stress produced by the process of extrusion. The results of these and other studies support the possibility that the presence of an intact and successfully expanded cumulus mass aids in oocyte maturation, optimal ovulation and fertilizability [26], oocyte transport to the oviduct [33], the zona reaction [34], sperm motility [35–37], sperm attraction [38], sperm selection [39, 40], and the acrosome reaction [41].
To further investigate the function(s) of cumulus expansion within intact animals, we designed experiments to examine the effect(s) of competitively inhibiting the direct binding of II and endogenous HA during the preovulatory period in hormonally primed mice. We considered the possibility that injection of excess HA oligosaccharides into hormonally primed animals might result in sloughing of the cumulus from COCs while the COCs were still resident within the intact responding follicle. Our primary interest was to assess the effect of cumulus cell disaggregation in vivo on COC morphology, ovulation, fertilization, and viability of the oocyte.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immature female (20 days old), adult female (5–6 wk old), and adult male proven breeder ICR mice were purchased from Harlan-Sprague Dawley (Indianapolis, IN). Mice were housed in micro-isolator cages in rooms with constant humidity and temperature. The animals were fed standard chow and water while exposed to a 12L:12D dark cycle.
Materials
Equine CG was purchased from Professional Compounding Centers of America Inc. (Houston, TX). Chondroitin sulfate C, hCG, HA (from rooster combs), hyaluronidase from bovine testes, Streptomyces hyaluronidase, porcine FSH, D-(+)-glucosamine, penicillin-G, DL-lactic acid, and mineral oil were purchased from Sigma Chemical Company (St. Louis, MO). Minimal Essential Medium (MEM) was purchased from Life Technologies, Inc. (Gaithersburg, MD). Streptomycin sulfate was purchased from Grand Island Biological Co. (Grand Island, NY). Disposable tissue culture filters were purchased from Nalgene Co. (Rochester, NY). Prestained molecular weight markers were purchased from Bio-Rad Laboratories (Hercules, CA). The ECL Western blotting detection reagents and Hyperfilm autoradiography film were purchased from Amersham Life Science (Arlington Heights, IL). Peroxidase-conjugated AffiniPure F(ab')2 fragment donkey anti-rabbit IgG (heavy and light chains) and fluorescein isothiocyanate (FITC)-conjugated AffiniPure F(ab')2 fragment goat anti-rabbit IgG (heavy and light chains) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Affinity-purified rabbit anti-human II IgG was purchased from Dako (Carpenteria, CA).
HA Oligosaccharides
Previous studies using the methods of Hascall and Heinegard [42] and Holmbeck and Lerner [43], with minor modifications, have shown that a solution of HA oligosaccharides (5–100 disaccharides) can be produced by partial degradation of rooster comb HA with testicular hyaluronidase. After heat inactivation of the enzyme, the oligosaccharide solution was sterile-filtered twice using a 0.2-µm Nalgene disposable tissue culture filter. Parallel experiments determined the sizes of the oligosaccharides by separating the digested mixture using a DEAE ion exchange column and a quantitative carbazole-based assay of HA with a reducing power assay (data not shown; [43, 44]). To ensure that a substantial amount of HA oligosaccharides would be present at the time of II and HA binding (~6 h post-hCG), the half-life of the oligosaccharides was quantitated using the Bitter and Muir [45] carbazole assay. Briefly, blood was drawn from the animals at varying time points between 5 and 20 h post-hCG and HA oligosaccharide administration. Uronic acid levels were analyzed, and the oligosaccharide half-life was deduced from a linear fitting of the exponential decay function. The changes in the total uronic acid concentration in the blood were plotted, and the half-life for the HA oligosaccharides was found to be 6.9 h (data not shown). Thus, a substantial amount of oligosaccharides would be present within the circulation and developing follicles to competitively inhibit the binding between II and endogenous HA.
In Vitro Cumulus Expansion
Immature female (20 days) ICR mice received injections of 5 IU eCG and were killed 48 h later. Ovaries were placed in MEM with antibiotics, and the COCs were isolated as described previously [14]. Approximately 15–20 compact preovulatory COCs were incubated in a 100-µl droplet of basic medium (MEM, 0.5 mg/ml D-glucosamine, 2.5 µg/ml pFSH, 0.4 µg/100 µl purified II) containing either 1) 0.4 µg/100 µl II, 2) 125–500 µg/100 µl chondroitin sulfate C, or 3) 60–200 µg/100 µl HA oligosaccharides under mineral oil. The COCs were incubated at 37°C in 5% CO2 for approximately 20 h, analyzed for stabilized/expanded cumulus masses, and photographed.
Animal Experiments
Folliculogenesis and ovulation were stimulated by priming all female animals with i.p injections of 5–10 IU eCG followed by 5–10 IU hCG (48 h later). All control animals used in the ovulation as well as the mating studies were treated with either PBS or chondroitin sulfate C (total of 15 mg in two injections of 150 µl PBS) via the tail vein as previously described [24]. All HA-treated animals used in the ovulation and mating studies received a total of 6 mg HA oligosaccharides 4 h and 6.5 h post-hCG via the tail vein. Each injection contained 3 mg HA oligosaccharides in 150 µl PBS. The effect of HA oligosaccharides on ovulation was assessed by harvesting oviducts from treated immature (20 days) animals 17 h post-hCG. The ovulated COCs were collected from the ampullar region, counted, and photographed. The effect of HA oligosaccharides on the developmental potential of the oocytes was assessed by placing treated adult females into separate cages containing one male either 6.5 h post-hCG or 18–20 h post-hCG. The presence of a vaginal plug 24 h later indicated successful mating. Approximately 35–40 h after mating, plugged females were removed, oviducts were harvested and flushed with PBS, and the resulting embryos were scored for developmental stages.
Rupture Site Visualization and Measurement
Ovulated COCs from hormonally primed immature animals treated with PBS, chondroitin sulfate C, or HA oligosaccharides were collected and measured using a 10 x 0.1-mm linear reticule. Ovaries from these animals were harvested, fixed with 4% paraformaldehyde, and washed with PBS. After the harvested ovaries were incubated in color development buffer (0.1 M Tris pH 9.5, 0.3 mg/ml nitroblue tetrazolium, and 0.5 mg/ml 5-bromo-4-chloro-3-indolyl phosphate) for approximately 15 min in the dark, rupture sites were visualized and measured with the 10 x 0.1-mm linear reticule.
Immunocytochemistry
Ovaries were harvested from HA-, PBS-, and chondroitin sulfate C-treated immature animals 7 h post-hCG. Ovaries were fixed in 4% paraformaldehyde and sectioned (8 µm) using a cryotome (Leitz, Wetzlar, Germany). Slides containing 15–20 ovary sections were incubated with rabbit anti-human II (1:1000) overnight at 4°C. Slides were washed in PBS, incubated with secondary antibody (1:1000, FITC:goat anti-rabbit IgG), observed with a Nikon fluorescence microscope (Nikon Corp., Melville, NY), and photographed.
Western Blot Analysis
Half of the samples, experimental and/or control, containing 15 ovulated COCs were treated with Streptomyces hyaluronidase for 3 h at 37°C. This enzyme is specific for HA, and digestion will release any II, PI, or heavy chains that are covalently bound to the HA contained within the cECM. Undigested and digested COCs and prestained molecular weight standards (Bio-Rad) were placed in sample buffer (125 mM Tris pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol), heated to 100°C for 90 sec, and resolved using 7% SDS-PAGE. After proteins were transferred to nitrocellulose, the membrane was probed with a rabbit anti-human polyclonal antibody specific for both the light and heavy chains of II (1:500). Detection of II specific epitopes was achieved by incubating the probed membrane in peroxidase-conjugated AffiniPure F(ab')2 fragment donkey anti-rabbit IgG (heavy and light chains; 1:60 000) and developed using an ECL kit according to the manufacturer's protocol.
Statistical Analysis
Rupture site and COC diameter data were analyzed using a Kruskal-Wallis one-way ANOVA of ranks followed by pair-wise multiple-comparison procedures using Dunn's method; p 
0.0001. Ovulation data were analyzed using one-way repeated-measures ANOVA followed by pair-wise multiple-comparison procedures (Student-Neuman-Keuls, Dunn's, or Bonferroni's method; p 0.0001). Mating data were analyzed using Fisher's contingency table followed by a modified Tukey-like test with arc sine transformation of the original data (p 0.001).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because of its large average size (Mr 2 000 000) and high viscosity, whole molecules of the glycosaminoglycan HA could not be successfully injected directly into the vasculature. Therefore, intact HA was digested with hyaluronidase to yield smaller oligosaccharide (20–100 disaccharide) subunits. Previous studies have shown that oligosaccharides larger than five disaccharide subunits disrupt in vitro cumulus expansion [25]. However, the source of the stabilizing II proteins in those experiments was fetal bovine serum (FBS). To ensure that the HA oligosaccharides specifically interrupted the binding between the critical stabilizing components of the cECM (II and HA), we used purified II instead of FBS to stabilize the cultured COCs in preliminary expansion studies. Furthermore, we chose to use another glycosaminoglycan, chondroitin sulfate C, in addition to PBS as a control reagent for the following reasons: 1) it is small in size (~62 kDa) and able to permeate the blood follicle barrier before and after an ovulatory surge; 2) it lacks the extreme viscosity of native HA and can therefore be directly injected into the vasculature; 3) it possesses a long half-life when administered to laboratory animals [46–48]; and 4) previous in vitro studies using either FBS or purified II/PI established that this glycosaminoglycan does not disrupt the cumulus in cultured COCs, even at high concentrations (up to 5 mg/ml; [25]).
When COCs were cultured in the presence of purified II and chondroitin sulfate C or purified II alone, expansion and stability of the cECM was normal (Fig. 1, A and B, respectively). In contrast, the presence of HA oligosaccharides in culture media containing II resulted in the destabilization of the expanded cumulus mass (Fig. 1C). Thus, the HA oligosaccharides were found to possess the same disruptive attributes as intact HA molecules, presumably through the competitive inhibition of the binding between II and endogenous HA.
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Disruption of In Vivo Cumulus Expansion Decreased Ovulation Rates
After determining that HA oligosaccharides successfully disrupted in vitro cumulus expansion, our next goal was to disrupt cumulus expansion in vivo by administering HA oligosaccharides to hormonally primed immature animals. The injection of HA oligosaccharides had no observable effects on the health or behavior of the mice. However, the administration of HA oligosaccharides during the preovulatory period had profound effects on the morphology and composition of the COCs and on the rate of ovulation. COCs obtained from the oviducts of animals treated with HA oligosaccharides (Fig. 2, left) were smaller and only loosely associated with one another, unlike COCs obtained from the oviducts of animals injected with PBS (Fig. 2, right) or with chondroitin sulfate C (data not shown). Although the diameters of the COCs from HA oligosaccharide-treated animals were significantly smaller than those from controls, the rupture sites created during the process of ovulation were similar in the PBS-, chondroitin sulfate C-, and HA oligosaccharide-treated animals (Fig. 3). In addition, the injection of HA oligosaccharides significantly reduced the number of ovulated COCs compared to those in controls (Fig. 4).
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HA Oligosaccharides Decreased the Amount of II Incorporated In Vivo
To determine whether the reductions in COC size and number resulted from a decrease in the amount of II incorporated into the cECM, immunocytochemical and Western blot analyses were performed using polyclonal antibodies specific for II. Immunocytochemistry of ovarian sections demonstrated that HA oligosaccharide treatment of hormonally primed animals decreased the amount of II incorporated into the expanding cECM within developing follicles compared to PBS- or chondroitin sulfate C-treated animals (Fig. 5). Moreover, Western blot analysis of isolated COCs exhibited a dramatic decrease in not only the amount but also the molecular configuration of II subunits present within the cECM (Fig. 6). The content of the cECMs was analyzed by digesting ovulated COCs from control and HA-treated animals with Streptomyces hyaluronidase. If the COCs were not digested (Fig. 6, lanes 2, 4, 6), the large complexes of HA and linked proteins did not enter the gel upon electrophoresis. Thus, COCs from chondroitin sulfate C- or PBS-treated animals were found to contain both II double heavy chains (~160 kDa) and single heavy chains (~85–115 kDa; Fig. 6, lanes 3 and 5). In contrast, COCs from HA treated-animals had a reduced amount of II single heavy chain and no discernable double heavy chains (Fig. 6, lane 7).
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A Disrupted Cumulus Mass Decreased Oocyte Viability
Although the cumulus masses produced within HA oligosaccharide-treated animals were morphologically smaller and irregular compared to those of controls, the oocytes within the masses appeared normal in size and shape (Fig. 2, arrows). To determine whether these oocytes possessed the same viability and developmental potential as those surrounded by intact and optimally expanded cumulus masses, experimental and control animals were mated either before (6.5 h post-hCG) or after (18 h post-hCG) ovulation. Because mice begin to ovulate 12 h after an ovulatory stimulus, mating 6.5 h after administration of hCG resulted in immediate fertilization since sperm were already present within the oviduct at the time COCs were extruded from the follicle. In contrast, in animals mated 18–20 h after administration of hCG, fertilization of the ovulated COCs occurred after they had been retained within the oviduct for 6–8 h. After the adult animals were mated, the oviducts were flushed, and the resulting morphologically normal embryos (2–4 cells) or fragmented oocytes/embryos were counted. Mating before ovulation resulted in no significant differences in the proportions of 1-cell, 2- to 4-cell, or fragmented embryos in experimental and control animals (Table 1). However, as shown in Table 1, when animals were mated after ovulation, a significant decrease in the percentage of 2- to 4-cell embryos in HA oligosaccharide-treated animals (61.1%) was apparent compared to PBS (79.0%)- or chondroitin sulfate C (76.3%)-treated animals. Conversely, the percentage of fragmented oocytes/embryos in the HA oligosaccharide-treated animals was significantly higher (30.4%) than in the PBS (13.25%)- or chondroitin sulfate C (16.4%)-treated animals. Thus, the developmental potential and viability of oocytes surrounded by a destabilized and morphologically abnormal cumulus mass were compromised during the process of fertilization and early cleavage.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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I/PI and Cumulus Synthesized HA Disrupts the Process of Cumulus Expansion
Upon initial entry into follicles after an ovulatory stimulus, native PI and II are bound to cumulus-generated HA through a charge-mediated interaction. Further stabilization of the cECM begins 6 h after an ovulatory stimulus, when factor(s) produced by the granulosa cells catalyze a conversion from this charge-mediated interaction to a covalent binding between the heavy chains and HA. Chen et al. [21] and Zhao et al. [49] have determined that this binding occurs via an aspartic acid moiety at the carboxyl terminus of the heavy chains. Once the COCs are stabilized, however, the covalently bound heavy chains can be cleaved from the HA through treatment with hyaluronidase and revealed on Western blots as single or double heavy-chain bands (~85 kDa and ~160 kDa, respectively, [21]). Upon hyaluronidase treatment, COCs produced by animals treated with HA oligosaccharides were found to possess only single heavy chains (Fig. 6). Since the double heavy chains are thought to be produced by the binding of HA to II rather than to PI [21], it is possible that the exogenous HA oligosaccharides may have interfered more completely with II-HA binding than with PI-HA binding.
Chondroitin sulfate C had little or no effect on the incorporation of II/PI into COCs. This result is consistent with a mechanism of heavy chain-HA binding that results in a concomitant loss of the chondroitin sulfate-bikunin moiety from the same site, possibly through a transesterification reaction [21, 49].
Disruption of Cumulus Expansion by Exogenous HA Oligosaccharides May Directly Affect the Process of Ovulation
Because the HA oligosaccharide-treated animals produced COCs similar in size to their rupture sites, it is possible that these smaller COCs were not as proficient as fully expanded COCs in "plugging" the ovarian rupture sites. Therefore, proper extrusion of these COCs may have been affected because a constant intrafollicular pressure could not be maintained during the preovulatory period. This may also explain the complete inhibition of ovulation in DON-injected mice, in which the diameter of the COCs is never greater than about 100 µm [26].
In addition to the reduced size of the ovulated COCs, we noted a modification in their physical quality. HA-treated animals produced COCs that were not "sticky" and therefore did not clump within the ovarian ampulla as did ovulated COCs from control follicles. Previous studies have demonstrated that the mode of II/PI binding is related to the physical quality of the cumulus mass. For example, Western blot analysis has shown a significant difference in the mechanism by which II and PI are bound to HA during in vitro and in vivo maturation assays. Only the initial step in II/PI binding occurs in the cultured COCs, whereas the heavy chains alone (after cleavage and removal of the chondroitin sulfate-bikunin moiety) become covalently bound to HA in the in vivo-matured COCs. Additionally, when trituration assays were performed with pipette bores half the diameter of COCs, those matured in vivo were found to have a greater resistance to shear force compared to in vitro-matured COCs (unpublished results). Likewise, the resistance of the cECM to environmental stresses such as acid pH and high ionic strength were found to be greater in the in vivo-matured COCs [15]. Thus, it is the differences in the II/PI or heavy chain mode of binding to HA that appear to account for differences in viscoelastic properties and sensitivity to acid and ions during these different maturation conditions. The significance of the absence of double heavy chains in HA oligosaccharide-treated animals is not understood. However, it is possible that the reduction in HA-bound single heavy chains or the lack of HA-bound double heavy chains in the cECM of HA oligosaccharide-treated animals may contribute directly or indirectly to the modification of their physical character, thus interfering with their extrusion from the follicle at ovulation.
In contrast to the effects of HA oligosaccharides, the administration of chondroitin sulfate C did not have a significant effect on the rate of ovulation (Fig. 4). This result is consistent with the loss of chondroitin sulfate from the cumulus mass during cumulus expansion rather than its direct participation in cECM stabilization.
Disruption of Cumulus Expansion Affects the Development of COCs Stored Within the Oviduct
Ovulated COCs from HA-treated animals possessed a smaller cumulus mass, but the enclosed oocyte did not appear to have any significant morphological abnormalities when compared to control COCs (Fig. 2). In comparisons of embryos obtained from control mice or HA oligosaccharide-treated mice inseminated 6.5 h after hCG, there was no significant difference in the proportion of embryos that advanced to the 2-cell stage and/or were fragmented. These data support the idea that alteration of the size or composition of the COCs by excess HA did not, in itself, affect the rate of fertilization or early cleavage (Table 1). Moreover, since the proportion of fragmented oocytes/embryos was virtually identical in all three groups, the hypothesized protective functions of abnormal or intact cumulus masses cannot be discriminated in these animals.
In contrast to these results, significant differences were observed in the proportion of embryos developing to the 2- to 4-cell stage as well as in the proportion of fragmented embryos when treated mice were inseminated 6–8 h after ovulation. Assuming that the fragmented embryos were fertilized, the overall fertilization rates were similar in HA oligosaccharide-treated and control animals. Therefore, it seems likely that storage of COCs with a disrupted cumulus mass in the oviducts had no effect on the rate of fertilization, but instead affected the early development of the embryos.
It has been established here and in previous studies that an intact cumulus mass protects developing oocytes before and after fertilization [25, 26, 33–41]. For example, Sato et al. [48] demonstrated that cumulus-produced component(s) (i.e., glycosaminoglycans) delay the degenerative process during in vitro oocyte culturing. Additionally, during cryopreservation, oocytes with an intact cumulus possess better fertilization rates after thawing than those frozen without cumulus cells [50]. As in the case of freezing, Edwards and Hanson [51] determined that detrimental effects (i.e., reduced oocyte protein synthesis) of heat shock on developing oocytes could be reduced by the presence of cumulus cells.
It is also possible that alterations in specific events involving sperm function during fertilization may have been responsible for the increased percentage of fragmentation in HA oligosaccharide-treated animals. For example, partially denuded oocytes appear to be more prone to polyspermy, which tends to result in embryo fragmentation [52]. Therefore, it has been suggested that the cumulus may play at least a passive role in egg penetrability in vitro by limiting the number of sperm reaching the zona. Additionally, the cumulus may act to select an optimal population of hypermotile and capacitated sperm during fertilization. As the sperm pass through the cECM, they rapidly complete the final stages of capacitation (via stimulation from cumulus components), enabling them to undergo the acrosome reaction when they arrive at the zona pellucida [53, 54]. Consequently, a destabilized cECM similar to those produced in these studies may not be adequate to fully capacitate sperm during fertilization. The smaller and less stabilized cumulus mass of COCs from HA-treated animals may be insufficient to limit the number of capacitated sperm reaching/penetrating the zona, thus resulting in polyspermy and eventually fragmentation.
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FOOTNOTES |
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2 Correspondence: William J. Larsen, Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati, College of Medicine, PO Box 670521, Cincinnati, OH 45267–0521. FAX: 51 558 4454; william.larsen{at}uc.edu
Accepted: March 19, 1999.
Received: January 26, 1999.
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R. Prochazka, P. Kalab, and E. Nagyova Epidermal Growth Factor-Receptor Tyrosine Kinase Activity Regulates Expansion of Porcine Oocyte-Cumulus Cell Complexes In Vitro Biol Reprod, March 1, 2003; 68(3): 797 - 803. [Abstract] [Full Text] [PDF]
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L. L. Espey and J. S. Richards Temporal and Spatial Patterns of Ovarian Gene Transcription Following an Ovulatory Dose of Gonadotropin in the Rat Biol Reprod, December 1, 2002; 67(6): 1662 - 1670. [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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 S. Varani, J. A. Elvin, C. Yan, J. DeMayo, F. J. DeMayo, H. F. Horton, M. C. Byrne, and M. M. Matzuk Knockout of Pentraxin 3, a Downstream Target of Growth Differentiation Factor-9, Causes Female Subfertility Mol. Endocrinol., June 1, 2002; 16(6): 1154 - 1167. [Abstract] [Full Text] [PDF]
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N. Kimura, Y. Konno, K. Miyoshi, H. Matsumoto, and E. Sato Expression of Hyaluronan Synthases and CD44 Messenger RNAs in Porcine Cumulus-Oocyte Complexes During In Vitro Maturation Biol Reprod, March 1, 2002; 66(3): 707 - 717. [Abstract] [Full Text] [PDF]
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A. J. Day and G. D. Prestwich Hyaluronan-binding Proteins: Tying Up the Giant J. Biol. Chem., February 8, 2002; 277(7): 4585 - 4588. [Full Text] [PDF]
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 J. S. Richards, D. L. Russell, S. Ochsner, M. Hsieh, K. H. Doyle, A. E. Falender, Y. K. Lo, and S. C. Sharma Novel Signaling Pathways That Control Ovarian Follicular Development, Ovulation, and Luteinization Recent Prog. Horm. Res., January 1, 2002; 57(1): 195 - 220. [Abstract] [Full Text] [PDF]
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M. D. Calder, A. N. Caveney, M. E. Westhusin, and A. J. Watson Cyclooxygenase-2 and Prostaglandin E2(PGE2) Receptor Messenger RNAs Are Affected by Bovine Oocyte Maturation Time and Cumulus-Oocyte Complex Quality, and PGE2 Induces Moderate Expansion of the Bovine Cumulus In Vitro Biol Reprod, July 1, 2001; 65(1): 135 - 140. [Abstract] [Full Text] [PDF]
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O. Carrette, R. V. Nemade, A. J. Day, A. Brickner, and W. J. Larsen TSG-6 Is Concentrated in the Extracellular Matrix of Mouse Cumulus Oocyte Complexes Through Hyaluronan and Inter-Alpha-Inhibitor Binding Biol Reprod, July 1, 2001; 65(1): 301 - 308. [Abstract] [Full Text] [PDF]
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S. Yoshioka, S. Ochsner, D. L. Russell, T. Ujioka, S. Fujii, J. S. Richards, and L. L. Espey Expression of Tumor Necrosis Factor-Stimulated Gene-6 in the Rat Ovary in Response to an Ovulatory Dose of Gonadotropin Endocrinology, November 1, 2000; 141(11): 4114 - 4119. [Abstract] [Full Text] [PDF]
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L. Zhuo, M. Yoneda, M. Zhao, W. Yingsung, N. Yoshida, Y. Kitagawa, K. Kawamura, T. Suzuki, and K. Kimata Defect in SHAP-Hyaluronan Complex Causes Severe Female Infertility. A STUDY BY INACTIVATION OF THE BIKUNIN GENE IN MICE J. Biol. Chem., March 9, 2001; 276(11): 7693 - 7696. [Abstract] [Full Text] [PDF]
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