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Biology of Reproduction 63, 1067-1074 (2000)
© 2000 Society for the Study of Reproduction, Inc.

ARTICLES

Effects of Midkine During In Vitro Maturation of Bovine Oocytes on Subsequent Developmental Competence1

Shuntaro Ikedaa, Keiko Ichihara-Tanakab, Tohru Azumaa, Takashi Muramatsub, and Masayasu Yamada2,,a

a Laboratory of Reproductive Physiology, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan b Department of Biochemistry, Nagoya University School of Medicine, Nagoya 466-8550, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Midkine (MK) is known to be a member of a new family of heparin-binding growth/differentiation factors, together with pleiotrophin, and to be quite rich in bovine follicular fluid. To examine whether treatment with MK during in vitro maturation (IVM) of bovine granulosa-enclosed oocytes affects their nuclear maturation and postfertilization development to the blastocyst stage, bovine granulosa-enclosed oocytes obtained from slaughterhouse-derived ovaries were cultured for 24 h in IVM medium without (control) or with various concentrations (1–500 ng/ml) of MK, followed by in vitro fertilization (IVF) and culturing. Although the MK treatment during IVM did not affect the rate of nuclear maturation or the postfertilization cleavage of oocytes, MK at >= 10 ng/ml significantly (P < 0.05) increased the blastocyst yields per tested and per cleaved oocyte compared with the case of the control. Next, the effects of various glycosaminoglycans (heparin, heparan sulfate, chondroitin sulfate A and C, and hyaluronic acid) preincubated with MK at 50 ng/ml were examined. The enhancing activity of MK was completely suppressed by heparin at 600 ng/ml but not by the other compounds. The effects of MK during IVM were also tested on oocytes freed from granulosa cells (GCs). When the denuded oocytes were cultured in IVM medium, no blastocyst formation after IVF was observed, regardless of MK supplementation. However, coculture of the denuded oocytes with isolated GC pellets enhanced the cleavage rates and the blastocyst yield, and these effects were more pronounced with MK supplementation. These results indicate that the presence of MK during IVM of bovine granulosa-enclosed oocytes can enhance their developmental competence to the blastocyst stage after IVF and suggest that the enhancing effects might be mainly mediated by GCs.

oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In in vitro maturation (IVM), fertilization (IVF), and culture (IVC) systems of bovine oocytes, the capacity for fertilization and embryonic development is influenced to a large extent by IVM conditions [1, 2]. Preovulatory mammalian oocytes, when removed from follicles, can spontaneously undergo meiotic maturation in culture, resulting in morphologically normal secondary oocytes arrested at the metaphase stage of the second meiotic division (MII) [3]. Although the spontaneous nuclear maturation of oocytes appears to occur normally in vitro, the functional normality of the oocytes for postfertilization development is apparently not completely normal because it is known that development of in vitro-matured bovine oocytes to the preimplantation stages following IVF is generally less successful than that of oocytes matured in vivo [4–6]. This is believed to be due to incomplete acquisition of developmental competence during oocyte maturation, and it seems likely that this acquisition is exclusively regulated by maturation events that occur in the cytoplasm rather than the nucleus of oocytes (these maturation events are referred to as cytoplasmic maturation and nuclear maturation, respectively) [2, 6, 7].

In vivo, both nuclear maturation and cytoplasmic maturation of preovulatory oocytes are mainly regulated by microenvironmental factors in follicles as follows: 1) follicular fluid containing steroids and growth factors [8] and 2) interaction between oocytes and follicular cells [8, 9], as well as by endocrine factors such as gonadotropins [10]. Attempts have been made to improve the IVM system for bovine oocytes, in some of which the effects of addition of follicular fluid to IVM media on oocyte maturation were examined [1, 11–14]. However, recent studies have shown that the efficacy of follicular fluid as a supplement to IVM medium to improve cytoplasmic maturation varies with its origin, dependent on such factors as follicular size [11, 12], follicular quality [12], time lapsed after LH surge [13], and ages of donors [14]. It has been widely suggested that follicular fluid contains not only positive but also negative factors that affect cytoplasmic maturation of oocytes in vitro, and the proportions of these factors are substantially changed with follicle status [12, 13]. Therefore, attempts have been made to determine what factor(s) in follicular fluid are responsible for cytoplasmic maturation of oocytes, which is usually evaluated by the ability of oocytes to develop to the blastocyst stage after IVF. In the search for such factors, the effects of various growth factors present in follicular fluid during IVM have been assessed, revealing that cytoplasmic maturation is partly regulated by epidermal growth factor (EGF) [15, 16], insulin-like growth factor-I (IGF-I) [17, 18], activin A [19, 20], and inhibin A [19]. However, the regulation of cytoplasmic maturation may not be mediated by only one specific growth factor in follicular fluid [14].

Recently, we found that when pooled follicular fluid was partitioned into heparin-adsorbed and -nonadsorbed fractions by heparin affinity chromatography and their effects on IVM of bovine oocytes were examined, the heparin-adsorbed fraction markedly enhanced the ability of oocytes to develop to the blastocyst stage after IVF in a dose-dependent manner, whereas the nonadsorbed fraction inhibited cytoplasmic maturation [21]. These findings prompted us to explore the possibility that one or more heparin-binding growth factors that exist in bovine follicular fluid might be involved in enhancing the cytoplasmic maturation. To our knowledge, the major heparin-binding growth factors in follicular fluid are basic fibroblast growth factor (bFGF) [22], midkine (MK), and pleiotrophin (PTN) [23]. However, bFGF has been shown not to enhance the acquisition of developmental competence when added to IVM medium of bovine oocytes [24].

On the other hand, MK and PTN, which constitute a new family of heparin-binding growth/differentiation factors [25], are quite rich in bovine follicular fluid at concentrations of 125 and 400 ng/ml, respectively [23]. Sequence analyses of MK and PTN showed that they have nearly 50% amino acid sequence homology and perfect conservation of 10 cysteine residues, which explains the observation that MK and PTN have redundant functions in both growth and differentiation in mammals, for example, neurite outgrowth activity [26]. It is also known that MK is produced by granulosa cells under the control of gonadotropins and might play a role in follicular growth [27, 28]. However, the roles of MK and PTN in oocyte maturation in vivo and in vitro have not yet been elucidated.

The present study was undertaken to examine the effects of MK at physiological concentrations during the IVM period of bovine granulosa-enclosed oocytes on postfertilization development, especially in terms of cleavage and blastocyst formation. In addition, to evaluate the mode of action of MK, oocytes stripped from the surrounding granulosa cells were also cultured in IVM medium with or without MK in the presence or absence of isolated granulosa cell pellets, and their developmental competence after IVF was examined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Recombinant Mouse MK

Recombinant mouse MK was produced by means of a baculovirus expression system as described previously [29].

Culture Media

The basal maturation medium (IVMM) used was synthetic oviduct fluid (SOF) described by Tervit et al. [30] with minor modifications (mSOF). It consisted of SOF, 2% (v/v) BME amino acids solution (Sigma Chemical Co., St. Louis, MO), 1% (v/v) MEM non-essential amino acids solution (Sigma), 0.5 mg/ml polyvinylalcohol (PVA; Sigma), 1 µg/ml estradiol-17ß (Sigma), and 100 IU/ml hCG (Sankyo Co., Tokyo, Japan).

The washing medium used for spermatozoa was modified Tyrode's balanced salt solution (BO, [31]) supplemented with 10 mM caffeine sodium benzoate (Sigma) and 20 µg/ml heparin (Sigma) (BO-1). The medium used for sperm preincubation and fertilization of oocytes was a 50:50 (v/v) mixture of BO-1 and BO supplemented with 20 mg/ml BSA (Sigma) (BO-2). Glucose was not added to BO because glucose was reported to delay capacitation of bovine sperm [32].

The embryo culture medium (IVCM) was similar to mSOF described above except for supplementation with 1% or 5% heat-inactivated fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS) with no PVA and hormones.

In Vitro Maturation

Ovaries collected from Japanese Black cattle at a local slaughterhouse were transported to the laboratory within 3 h in saline (9 g NaCl/L) at 35 to 38°C. The ovaries were pooled regardless of the stage of estrous cycle of the donors. Granulosa-enclosed oocytes were aspirated from 2- to 5-mm follicles. The oocytes with homogeneous and evenly granulated cytoplasm, which were surrounded by an intact granulosa cell mass were selected. Groups of 8 to 12 granulosa-enclosed oocytes or oocytes freed from surrounding granulosa cells by vortexing in PBS supplemented with 0.5 mg/ml PVA (denuded oocytes; DO) were randomly allocated into different treatment groups, transferred to 50-µl drops of medium for IVM covered with paraffin oil and then cultured for 24 h at 39°C in 5% CO2 in air.

In Vitro Fertilization and In Vitro Culture

Two straws (0.5 ml/straw) of frozen bull semen from a single ejaculate were thawed quickly in a water bath at 38°C. Spermatozoa were washed twice with BO-1 by centrifugation at 500 x g for 3 min. Sperm pellets were resuspended at a concentration of 4 to 5 x 106 sperm/ml in BO-2. The sperm suspensions in 100-µl drops were placed onto a plastic dish (Nunc, Roskilde, Denmark), covered with prewarmed paraffin oil, and incubated for 40 min at 39°C under 5% CO2 in air before insemination. Groups of 8 to 12 presumptive matured oocytes with surrounding granulosa cells or DOs were placed in the 100 µl of sperm suspensions. Gametes were coincubated for 6 h at 39°C under 5% CO2 in air. With these procedures, the rates of polyspermic fertilization and parthenogenesis were less than 15% and 5% of oocytes, respectively.

After 6 h, oocytes except DOs were freed from surrounding granulosa cells by repeated pipetting through a fine-bored pipette. The presumptive zygotes were transferred into 50-µl drops of IVCM containing 1% FBS and cultured at 39°C under 5% CO2, 5% O2, and 90% N2. At 48 h postinsemination, the morphologically normal cleaved embryos were transferred to newly prepared IVCM containing 5% FBS and then cultured until Day 8 (fertilization = Day 0) at 39°C under 5% CO2, 5% O2, and 90% N2.

Experimental Designs

In each experiment, treatments were performed only during the IVM period.

Experiment 1: Effects of MK on nuclear maturation and postfertilization development Granulosa-enclosed oocytes were treated with MK at 0 (control), 1, 10, 50, 100, and 500 ng/ml. These concentrations were chosen on the basis of the physiological concentration of MK in bovine follicular fluid described above [23]. To evaluate the nuclear maturation rates, 24 h after commencement of IVM, some oocytes were freed from granulosa cells by pipetting, fixed with ethanol-acetic acid (3:1), stained with 1% orcein, and assessed by phase-contrast microscopy (x400). Maturation was indicated by the presence of both contracted MII chromosomes and smaller polar body chromosomes. The remaining oocytes were subjected to the IVF and IVC procedures described above. The cleavage rates were assessed on Day 2 of IVC (48 h postinsemination). On Days 7 and 8 of IVC (IVF = Day 0), the percentages of embryos reaching the blastocyst stage were recorded.

Experiment 2: Effects of heparin on MK action MK is a heparin-binding protein and is thought to be associated in vivo with a heparin-like chain in a heparan sulfate carbohydrate moiety on the cell surface [29, 33]. Therefore, we examined whether the effect of MK on granulosa-enclosed oocytes would be neutralized with heparin as a putative antagonist of heparan sulfate. Before the treatments, MK at 10 µg/ml was first preincubated with various concentrations (0, 40, 80, or 120 µg/ml) of heparin (Sigma) in 5 µl of IVMM overnight at 4°C. After the preincubation, mixtures of MK and various concentrations of heparin were diluted with IVMM to a final volume of 1 ml. Consequently, the final concentration of MK was adjusted to 50 ng/ml and that of heparin, to 0, 200, 400 or 600 ng/ml, respectively.

Granulosa-enclosed oocytes were treated in the respective diluted IVMM mixtures, followed by IVF and IVC as described above. The postfertilization development was assessed in terms of cleavage on Day 2 and blastocyst formation by Day 8.

Experiment 3: Effects of other glycosaminoglycans on MK action MK at 10 µg/ml was preincubated with each of 120 µg/ml heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, and hyaluronic acid (Seikagaku Corporation, Tokyo, Japan) in 5 µl of IVMM overnight at 4°C. After the preincubation, the mixtures of MK and each glycosaminoglycan were diluted with IVMM to a final volume of 1 ml. The final concentration of MK was 50 ng/ml, and that of each glycosaminoglycan was 600 ng/ml. Oocyte treatments and developmental assessment of oocytes were performed as in experiment 2.

Experiment 4: Effect of MK in the presence or absence of isolated granulosa cell pellet during IVM of DOs on their postfertilization development To assess whether the effect of MK on postfertilization development was due to a direct action on oocytes or to an action mediated via the granulosa cells (GCs) surrounding oocytes, DOs were cultured in IVMM with or without 50 ng/ml MK and in the presence or absence of GC pellet (see Fig. 1). For preparation of GC pellets, GCs obtained from small follicles at oocyte collection were washed twice with PBS and centrifuged at 1000 x g for 5 min. Pelleted GCs were washed once with IVMM and centrifuged at 1000 x g for 5 min. After the supernatant was discarded, the remaining GC pellet was carefully removed, leaving the cells in clumps, and then put into 50-µl drops of IVMM. The total number of GCs per drop was adjusted to 7.0 x 106. The developmental assessment of oocytes after IVM, IVF, and IVC was performed as in experiment 2.



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  		FIG. 1. Bovine granulosa-enclosed oocytes (A), DOs (B), and DOs with pelleted GCs (C) at the beginning of IVM culture. Bar = 150 µm

Statistical Analysis

Statistical analyses were performed using Stat View and Super ANOVA programs (Abacus Concepts Inc., Berkeley, CA). Raw percentage data of each replicate in each experiment were arcsine transformed to correct the deviation from the normal distribution. In experiments 1, 2, and 3, the arcsine-transformed data were subjected to one-factor ANOVA, and Fisher's PLSD test was used to detect significant differences among the treatments. In experiment 4, the arcsine-transformed data were subjected to two-factor ANOVA to detect the overall effect of each factor (presence or absence of MK and GCs) and interaction between them. In addition, contrasts procedure was used to detect significant differences among the 2 x 2 factorial groups. Data were expressed as mean values ± SEM. Significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1

As shown in Table 1, when granulosa-enclosed oocytes were cultured in the presence or absence of MK during IVM, the rates of oocytes reaching MII were not significantly different between IVMM with no MK (control; 78%) and IVMM with MK at any concentration (70–86%). The presence or absence of MK during IVM did not affect the cleavage rate (71–84%), either. However, the presence of MK at 10 to 500 ng/ml during IVM significantly (P < 0.05) increased the percentage of blastocysts on Day 7 (28–33%) compared with that for the control (16%). The rates of both total oocytes and cleaved oocytes that reached the blastocyst stage by Day 8 were also higher in the oocytes treated with MK at 10 to 500 ng/ml than in the control, and these rates were comparable to those (36% from total oocytes and 55% from cleaved oocytes, n = 107) of oocytes matured in IVMM with 10% FBS. However, the effects of 100 ng/ml MK treatment were not significant compared with those of the control (P = 0.07 and P = 0.06, respectively). MK at 1 ng/ml had no effect on the blastocyst yield.


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  		TABLE 1. Effect of MK during IVM on nuclear maturation and postfertilization development of bovine oocytes

Experiment 2

To ascertain whether the enhancing effects of MK on developmental competence of oocytes after IVF are mediated by the interaction with heparin-like moieties on the cell surface, the effect of heparin on MK action was examined (Fig. 2). In accordance with the results shown in Table 1, MK at 50 ng/ml did not affect the cleavage rate (control vs. MK treatment: 76.9 vs. 76.5%) but significantly (P < 0.05) increased the rate of blastocyst formation by Day 8 (33.6%) compared with that for the control (21.4%). When granulosa-enclosed oocytes were treated with heparin alone at 200, 400, and 600 ng/ml during IVM, postfertilization development to the cleavage (78.4%, 72.0%, and 71.1%, respectively) and the blastocyst stages (21.6%, 19.7%, and 21.1%, respectively) were not affected. On the other hand, in the presence of 50 ng/ml MK, heparin had no effect on the cleavage rates (68.1%, 69.7%, and 75.0% at 200, 400, and 600 ng/ml, respectively). However, the rates of oocytes that developed to blastocysts by Day 8 after IVF were decreased by heparin treatment in a dose-dependent manner, and at a concentration of 600 ng/ml, the rate was suppressed to the level of the control and heparin-alone treatment groups.



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  		FIG. 2. Effects of heparin on enhancing activity of MK during IVM on developmental competence of bovine oocytes after IVF. Data are presented as percentages of a total of 404 oocytes in four replicates. The number of oocytes tested per group is indicated in parentheses. The values with different superscripts differ significantly (P < 0.05)

Experiment 3

To clarify the specificity of the neutralizing effect of heparin on MK action shown in the above experiment, the effects of other glycosaminoglycans on MK action were examined (Fig. 3). Like heparin, the other glycosaminoglycans (heparan sulfate, chondroitin sulfate A and C, and hyaluronic acid) did not affect the cleavage rates after IVF (69.5, 72.5, 75.0, and 81.4%, respectively). However, the effects of these glycosaminoglycans were different from that of heparin with respect to the blastocyst formation: MK could exert its enhancing effect on blastocyst formation despite preincubation with these glycosaminoglycans (30.5, 34.1, 32.5, and 36.9% for heparan sulfate, chondroitin sulfate A and C, and hyaluronic acid, respectively) at a rate similar to that observed with treatment with MK alone (33.6%).



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  		FIG. 3. Effects of glycosaminoglycans other than heparin on developmental-competence-enhancing activity of MK during IVM. Data are presented as percentages of a total of 323 oocytes in four replicates. The numbers with each column represent the number of oocytes tested per group. Columns with different superscripts differ significantly (P < 0.05)

Experiment 4

To examine whether the presence of GCs that surround oocytes is required for the enhancing effects of MK on developmental competence of oocytes after IVF, DOs were treated with 50 ng/ml MK during IVM in the presence or absence of a GC pellet prepared by the method described in Materials and Methods. The percentages of embryos that developed to the cleavage and blastocyst stages in each treatment group are shown in Table 2, and the overall effects of MK and GC during IVM upon each developmental rate and the interaction between the two factors (MK and GC effects) are shown in Table 3. Overall, the presence or absence of GCs (GC+ or -) significantly affected the cleavage rates (P = 0.011). The presence or absence of MK (MK+ or -) overall did not exert a significant effect on the cleavage (P = 0.052). The highest cleavage rate was achieved in the MK+/GC+ group (34.9%), but it was not significantly different from that in the MK-/GC+ group (17.8%). The interaction between MK and GC effects on cleavage rates was not significant (P = 0.55).


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  		TABLE 2. Effect of MK in combination with the presence or absence of granulosa cell pellets (GC) during IVM of bovine DOs on postfertilization development


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  		TABLE 3. Two-factor ANOVA of MK and GC effects and their interaction.a

Both (MK+ or -) and (GC+ or -) overall significantly affected the rates of total oocytes and cleaved oocytes that developed to the blastocyst stage by Day 8 (MK: P = 0.0004 and 0.002, respectively; GC: P < 0.0001 and P < 0.0001, respectively). The absence of GCs resulted in no blastocyst yield, regardless of the presence or absence of MK. In the MK-/GC+ group, blastocysts were obtained, but the rates were very low (1.0% from total oocytes and 2.9% from cleaved oocytes). The beneficial effect of GCs was more pronounced with MK supplementation, and MK combined with GCs resulted in significantly (P < 0.0001) higher yield compared with the other factorial combinations. The interactions between MK and GC effects on the blastocyst yields (both from total and cleaved oocytes) were significant (P = 0.0004 and 0.002, respectively).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we first demonstrated that although treatment of bovine granulosa-enclosed oocytes with MK at physiological concentrations during the IVM period did not affect nuclear maturation or cleavage after IVF, the treatment was capable of stimulating the acquisition of in vitro developmental competence to the blastocyst stage. This finding suggests that MK has an ability to promote cytoplasmic maturation of oocytes, and it is supported by previous reports which suggested the presence of an enhancing activity for cytoplasmic maturation of bovine oocytes in follicular fluid [1, 11–13], particularly by our previous results that indicated that the enhancing activity of bovine follicular fluid could be partitioned to the heparin-adsorbed fraction by heparin affinity column chromatography [21]. Moreover, we have confirmed that the heparin-adsorbed fraction of bovine follicular fluid contains MK at a higher concentration than whole follicular fluid or the nonadsorbed fraction (unpublished results).

Previously, the enhancing effects of bovine follicular fluid on oocyte maturation have been attributed to various growth factors such as EGF [15, 16], IGF-I [17, 18], activin A [19, 20], and inhibin A [19]. Among these factors, EGF has been regarded as of major importance in various species (mouse [34]; rat [35]; pig [36]; cattle [15, 16, 24]). However, EGF appears to be responsible for nuclear maturation rather than cytoplasmic maturation of oocytes [15, 16, 24, 37]. In addition, Khatir et al. [14] pointed out that the maturation-promoting activity of follicular fluid for bovine oocytes cannot be completely explained by the presence of EGF because calf oocytes, which have the ability to respond to EGF, could not respond to follicular fluid, suggesting the possibility that another growth factor which remains to be identified could be involved. Regarding IGF-I and activin A, there are also some controversial reports (IGF-I [38]; activin A [39]). However, to our knowledge, there have been no previous reports in which MK was shown to have the ability to enhance cytoplasmic maturation of bovine oocytes.

MK was identified in bovine follicular fluid using an assay that detected growth-promoting activity in bovine aortic smooth muscle cells, and the concentration of MK in bovine follicular fluid was estimated to be 125 ng/ml [23]. This concentration is comparable to those of IGF-I [40], activin A [41], and inhibin A [42] in bovine follicular fluid and is much higher than that of EGF in porcine [43] and human [44] follicular fluid. Moreover, Minegishi et al. and Karino et al. reported that MK mRNA in immature rat ovary was located exclusively in granulosa cells of healthy follicles based on in situ hybridization and that the expression of MK mRNA was increased by eCG injection and decreased by eCG and hCG injection; this fluctuation pattern was similar to that of the FSH receptor mRNA level in the ovary [27, 28]. They also found that the MK mRNA level increased in a time- and dose-dependent manner in the presence of FSH in cultured rat granulosa cells [28]. These findings documented that MK expression in granulosa cells is regulated by gonadotropins and suggested that MK, produced under the control of gonadotropins, may play some functional role in the follicles, such as promotion of oocyte maturation. Taken together, the present and previous results suggest that MK is a physiological regulator that enhances cytoplasmic maturation of bovine oocytes.

Several heparin-binding growth factors, such as acidic and basic fibroblast growth factors, hepatocyte growth factor, and platelet-derived growth factor, have been reported to associate with the cell surface heparan sulfate chain of proteoglycans [45]. Likewise, it was reported that the neurite outgrowth activity of MK in culture was inhibited by addition of heparin as a putative antagonist of heparan sulfate on the nerve cell surface [29, 33] or by heparitinase digestion of target cells [29]. The present results, shown in Figures 1 and 2, demonstrated that the enhancing effect of MK during IVM on the developmental competence of oocytes to the blastocyst stage was strongly inhibited by low concentrations (400 to 600 ng/ml) of heparin but not by other glycosaminoglycans. Treatments with heparin alone during the IVM period at 200 to 600 ng/ml had no effect on the developmental competence of oocytes. Therefore, these data suggest that the heparin-like moiety of the heparan sulfate chain on bovine granulosa-oocyte complexes is required for MK to exert its effects. In several mammalian species, granulosa cells have been demonstrated to have heparan sulfate proteoglycan (HSPG) on their cell surface and extracellular matrix [46, 47]. It is interesting that heparan sulfate had no inhibitory effect on MK actions for neurite outgrowth activity [29] or the enhancing activity for cytoplasmic maturation of oocytes shown here. Kaneda et al. [29] proposed that the highly sulfated heparin-like structure required for MK action on embryonic neurons is unique and distinct from commercially available heparan sulfate purified from bovine kidney. Furthermore, oversulfated chondroitin sulfate chain, not the conventional one used in the present study, might be also involved in MK binding as suggested elsewhere [48].

The results of the experiments using DOs that are shown in Table 2 suggested that the enhancing effect of MK on developmental competence of granulosa-enclosed oocytes may be mediated by GCs, rather than by a direct action on oocytes. The developmental competence of DOs after IVF was markedly reduced compared with that of oocytes enclosed by GCs, irrespective of the presence or absence of MK. This observation is consistent with previous reports indicating the importance of follicular cells during bovine oocyte maturation [49, 50]. In fact, when DOs were cocultured with isolated GC pellets during IVM, the developmental ability of oocytes was recovered, and moreover, this ability was greater when DOs were treated with both GC pellets and MK. These findings suggest that oocyte maturation, especially cytoplasmic maturation, is regulated by a soluble factor(s) from GCs. However, taking the slightly increased cleavage rates of noncocultured DOs treated with MK into account, we cannot exclude the possibility that the positive effects of MK might partially be due to its direct action on oocytes. In addition, the fact that DOs could not be endowed with developmental competence equivalent to that of granulosa-enclosed oocytes, even with MK treatment, suggests that the contact of oocytes with GCs is also crucial for oocytes to efficiently acquire developmental competence.

In conclusion, our results indicate that MK treatment of bovine granulosa-enclosed oocytes during IVM can enhance the developmental competence following IVF, and they suggest that the effects of MK are mainly mediated by GCs. Further studies will be needed for evaluating the quality of blastocysts produced in the presence or absence of MK during IVM, as shown by cell number, the ratio of inner cell mass to trophectodermal cells, and ultimately, the developmental competence to live calves after embryo transfer.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. K.S. Kim (Yonam College of Agriculture, Chonan-Si, Choongnam, Korea) for helpful instruction in bovine IVM-F-C. We would also like to express thanks to the staff at the Second Market of the Kyoto City Wholesale Market, especially to Mr. K. Naka and Mr. T. Ohnishi, for allowing us access to ovaries.


   FOOTNOTES
 
First decision: 26 April 2000.

1 This work was supported in part by grants-in-aid from the Ito Foundation and the Association of Livestock Technology (Japan). Back

2 Correspondence: Masayasu Yamada, Laboratory of Reproductive Physiology, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606–8502, Japan. FAX: 81 75 753 6329; yamada{at}jkans.jkans.kais.kyoto-u.ac.jp Back

Accepted: May 16, 2000.

Received: March 13, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gordon I. Laboratory Production of Cattle Embryos. New York: CAB International; 1994: 30–142
  2. Brackett BG, Zuelke KA. Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology 1993; 39:43–64[CrossRef]
  3. Edwards RG. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 1965; 208:349–351[CrossRef][Medline]
  4. Leibfried-Rutledge ML, Critser ES, Eyestone WH, Northey DL, First NL. Development potential of bovine oocytes matured in vitro or in vivo. Biol Reprod 1987; 36:376–383[Abstract]
  5. Van de Leemput EE, Vos PLAM, Zeinstra EC, Bevers MM, Van der Weijden GC, Dieleman SJ. Improved in vitro embryo development using in vitro matured oocytes from heifers superovulated with a controlled preovulatory LH surge. Theriogenology 1999; 52:335–349[CrossRef][Medline]
  6. Eppig JJ. Coordination of nuclear and cytoplasmic oocyte maturation in eutherian mammals. Reprod Fertil Dev 1996; 8:485–489[CrossRef][Medline]
  7. Sirard MA, Blondin P. Oocyte maturation and IVF in cattle. Anim Reprod Sci 1996; 42:417–426[CrossRef]
  8. Driancourt MA, Thuel B. Control of oocyte growth and maturation by follicular cells and molecules present in follicular fluid. A review. Reprod Nutr Dev 1998; 38:345–362
  9. Larsen WJ, Wert SE, Brunner GD. A dramatic loss of cumulus cell gap junctions is correlated with germinal vesicle breakdown in rat oocytes. Dev Biol 1986; 113:517–521[CrossRef][Medline]
  10. Eppig JJ, Downs SM. Chemical signals that regulate mammalian oocyte maturation. Biol Reprod 1984; 30:1–11[Abstract]
  11. Sirard MA, Roy F, Patrick B, Mermillod P, Guilbault LA. Origin of the follicular fluid added to the media during bovine IVM influences embryonic development. Theriogenology 1995; 44:85–94[CrossRef]
  12. Carolan C, Lonergan P, Monget P, Monniaux D, Mermillod P. Effect of follicle size and quality on the ability of follicular fluid to support cytoplasmic maturation of bovine oocytes. Mol Reprod Dev 1996; 43:477–483[CrossRef][Medline]
  13. Romero-Arredondo A, Seidel GE Jr. Effects of follicular fluid during in vitro maturation of bovine oocytes on in vitro fertilization and early embryonic development. Biol Reprod 1996; 55:1012–1016[Abstract]
  14. Khatir H, Carolan C, Lonergan P, Mermillod P. Characterization of calf follicular fluid and its ability to support cytoplasmic maturation of cow and calf oocytes. J Reprod Fertil 1997; 111:267–275[Abstract/Free Full Text]
  15. Harper KM, Brackett BG. Bovine blastocyst development after in vitro maturation in a defined medium with epidermal growth factor and low concentrations of gonadotropins. Biol Reprod 1993; 48:409–416[Abstract]
  16. Lonergan P, Carolan C, Van-Langendonckt AV, Donnay I, Khatir H, Mermillod P. Role of epidermal growth factor in bovine oocyte maturation and preimplantation embryo development in vitro. Biol Reprod 1996; 54:1420–1429[Abstract]
  17. Harper KM, Brackett BG. Enhanced bovine oocyte quality after in vitro maturation (IVM) with insulin-like growth factor-I (IGF-I) and gonadotropins. Biol Reprod 1992; 46(suppl 1):67 (abstract 67)
  18. Artini PG, Battaglia C, D'Ambrogio G, Barreca A, Droghini F, Volpe A, Ganazzani AR. Relationship between human oocyte maturity, fertilization and follicular fluid growth factors. Hum Reprod 1994; 9:902–906[Abstract/Free Full Text]
  19. Stock AE, Woodruff TK, Smith LC. Effects of inhibin A and activin A during in vitro maturation of bovine oocytes in hormone- and serum-free medium. Biol Reprod 1997; 56:1559–1564[Abstract]
  20. Silva CC, Knight PG. Modulatory actions of activin-A and follistatin on the developmental competence of in vitro-matured bovine oocytes. Biol Reprod 1998; 58:558–565[Abstract/Free Full Text]
  21. Ikeda S, Azuma T, Hashimoto S, Yamada M. In vitro maturation of bovine oocytes with fractions of bovine follicular fluid separated by heparin affinity chromatography. J Reprod Dev 1999; 45:397–404[CrossRef]
  22. Seli E, Zeyneloglu HB, Senturk LM, Bahtiyar OM, Olive DL, Arici A. Basic fibroblast growth factor: peritoneal and follicular fluid levels and its effect on early embryonic development. Fertil Steril 1998; 69:1145–1148[CrossRef][Medline]
  23. Ohyama Y, Miyamoto K, Minamino N, Matsuo H. Isolation and identification of midkine and pleiotrophin in bovine follicular fluid. Mol Cell Endocrinol 1994; 105:203–208[CrossRef][Medline]
  24. Kobayashi K, Yamashita S, Hoshi H. Influence of epidermal growth factor and transforming growth factor-alpha on in vitro maturation of cumulus cell-enclosed bovine oocytes in a defined medium. J Reprod Fertil 1994; 100:439–446[Abstract/Free Full Text]
  25. Kadomatsu K, Tomomura M, Muramatsu T. cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis. Biochem Biophys Res Commun 1988; 151:1312–1318[CrossRef][Medline]
  26. Muramatsu T. Midkine (MK), the product of a retinoic acid responsive gene, and pleiotrophin constitute a new protein family regulating growth and differentiation. Int J Dev Biol 1993; 37:183–188[Medline]
  27. Karino S, Minegishi T, Ohyama Y, Tano M, Nakamura K, Miyamoto K, Tanaka S, Ibuki Y. Regulation and localization of midkine in rat ovary. FEBS Lett 1995; 362:147–150[CrossRef][Medline]
  28. Minegishi T, Karino S, Tano M, Ibuki Y, Miyamoto K. Regulation of midkine messenger ribonucleic acid levels in cultured rat granulosa cells. Biochem Biophys Res Commun 1996; 229:799–805[CrossRef][Medline]
  29. Kaneda N, Talukder AH, Nishiyama H, Koizumi S, Muramatsu T. Midkine, a heparin-binding growth/differentiation factor, exhibits nerve cell adhesion and guidance activity for neurite outgrowth in vitro. J Biochem 1996; 119:1150–1156[Abstract/Free Full Text]
  30. Tervit HR, Whittingham DG, Rowson LE. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil 1972; 30:493–497[Abstract/Free Full Text]
  31. Brackett BG, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod 1975; 12:260–274[Abstract]
  32. Parrish JJ, Susko-Parrish JL, First NL. Capacitation of bovine sperm by heparin: inhibitory effect of glucose and role of intracellular pH. Biol Reprod 1989; 41:683–699[Abstract]
  33. Kaneda N, Talukder AH, Ishihara M, Hara S, Yoshida K, Muramatsu T. Structural characteristics of heparin-like domain required for interaction of midkine with embryonic neurons. Biochem Biophys Res Commun 1996; 220:108–112[CrossRef][Medline]
  34. Downs SM. Specificity of epidermal growth factor action on maturation of the murine oocyte and cumulus oophorus in vitro. Biol Reprod 1989; 41:371–379[Abstract]
  35. Dekel N, Sherizly I. Epidermal growth factor induces maturation of rat follicle enclosed oocytes. Endocrinology 1985; 116:406–409[Abstract/Free Full Text]
  36. Wang W, Niwa K. Synergetic effects of epidermal growth factor and gonadotropins on the cytoplasmic maturation of pig oocytes in a serum-free medium. Zygote 1995; 3:345–350[Medline]
  37. Kato H, Seidel GE Jr. Effects of EGF in bovine oocyte maturation medium on maturation in vitro fertilization and embryonic development. Theriogenology 1996; 45:276[CrossRef]
  38. Rieger D, Luciano AM, Modina S, Pocar P, Lauria A, Gandolfi F. The effects of epidermal growth factor and insulin-like growth factor I on the metabolic activity, nuclear maturation and subsequent development of cattle oocytes in vitro. J Reprod Fertil 1998; 112:123–130[Abstract/Free Full Text]
  39. Izadyar F, Zeinstra E, Colenbrander B, Vandertichele HMJ, Bevers MM. In vitro maturation of bovine oocytes in the presence of bovine activin A does not affect the number of embryos. Anim Reprod Sci 1996; 45:37–45[CrossRef][Medline]
  40. Spicer LJ, Echternkamp SE, Canning SF, Hammond JM. Relationship between concentrations of immunoreactive insulin-like growth factor-I in follicular fluid and various biochemical markers of differentiation in bovine antral follicles. Biol Reprod 1988; 39:573–580[Abstract]
  41. Knight PG, Muttukrishna S, Groom NP. Development and application of a two-site enzyme immunoassay for determination of "total" activin-A concentrations in serum and follicular fluid. J Endocrinol 1995; 148:267–279
  42. Guilbault LA, Rouillier P, Matton P, Glencross RG, Beard AJ, Knight PG. Relationships between the levels of atresia and inhibin contents (alpha subunit and alpha-beta dimer) in morphologically dominant follicles during their growing and regressing phases of development in cattle. Biol Reprod 1993; 48:268–273[Abstract]
  43. Hsu CJ, Holmes SD, Hammond JM. Ovarian epidermal growth factor activity, concentration in porcine follicular fluid during follicular enlargement. Biochem Biophys Res Commun 1987; 147:242–247[CrossRef][Medline]
  44. Hoffmann GE, Scott RT, Bryzski RG, Jones HW. Immunoreactive epidermal growth factor concentrations in follicular fluid obtained from in vitro fertilization. Fertil Steril 1990; 54:303–307[Medline]
  45. Ruoslahti E, Yamaguchi Y. Proteoglycans as modulators of growth factor activities. Cell 1991; 64:867–869[CrossRef][Medline]
  46. Yanagishita M, Mcquillan DJ. Two forms of plasma membrane-intercalated heparan sulfate proteoglycan in rat ovarian granulosa cells: labeling of proteoglycans with a photoactivatable hydrophobic probe and effect of the membrane anchor-specific phospholipase C. J Biol Chem 1989; 264:17551–17558[Abstract/Free Full Text]
  47. Drahorad J, Tesarik J, Cechjova D, Vilim V. Proteins and glycosaminoglycans in the intercellular matrix of the human cumulus-oophorus and their effect on conversion of proacrosin to acrosin. J Reprod Fertil 1991; 93:253–262[Abstract/Free Full Text]
  48. Maeda N, Ichihara-Tanaka K, Kimura T, Kadomatsu K, Muramatsu T, Noda M. A receptor-like protein-tyrosine phosphatase PT{zeta}/RPTPß binds a heparin-binding growth factor midkine: involvement of arginine 78 of midkine in the high affinity binding to PT{zeta}. J Biol Chem 1999; 274:12474–12479[Abstract/Free Full Text]
  49. Kim KS, Minami N, Yamada M, Utsumi K. Follicular cells affect the fertilizability and developmental competency of bovine oocytes in vitro. Reprod Fertil Dev 1997; 9:763–766[CrossRef][Medline]
  50. Zhang L, Jiang S, Wozniak PJ, Yang X, Godke RA. Cumulus cell function during bovine oocyte maturation, fertilization, and embryo development in vitro. Mol Reprod Dev 1995; 40:338–344[CrossRef][Medline]



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