Preovulatory follicular fluid during in vitro maturation decreases polyspermic fertilization of cumulus-intact porcine oocytes

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Theriogenology 70 (2008) 715–724 www.theriojournal.com

Preovulatory follicular fluid during in vitro maturation decreases polyspermic fertilization of cumulus-intact porcine oocytes In vitro maturation of porcine oocytes J. Bijttebier *, A. Van Soom, E. Meyer, B. Mateusen, D. Maes Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Received 13 February 2008; received in revised form 2 April 2008; accepted 24 April 2008

Abstract Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P < 0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos. # 2008 Elsevier Inc. All rights reserved. Keywords: Follicular fluid; Pig; In vitro maturation; Oocyte; Polyspermy

1. Introduction The ovarian follicle is the basic structural and functional unit of the mammalian ovary that provides the environment necessary for oocyte growth and maturation. Follicular fluid is composed of transudates from the serum through the blood–follicle barrier [1], but it also contains locally produced molecules. Supplementation of the oocyte maturation medium with 10%

* Corresponding author. Tel.: +32 9 264 7551; fax: +32 9 264 7797. E-mail address: [email protected] (J. Bijttebier). 0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.04.046

porcine follicular fluid (pFF) or serum is commonly practiced in current porcine in vitro fertilization (IVF) systems. Several studies have attempted to design chemically defined maturation media for pig oocytes that result in the production of blastocysts [2,3]. In spite of the development of these chemically defined media, pFF and/or serum supplementation is still superior which explains their overall use in in vitro production (IVP) systems of porcine embryos [4,5]. Moreover, piglet production after transfer of in vitro produced blastocysts has only been described after maturation of the oocytes in a medium supplemented with pFF, and recently, also after addition of fetal calf serum (FCS) [6–10]. The molecules

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that are responsible for the beneficial effects of pFF and serum are not identified yet, neither is the question resolved whether it concerns molecules which prevail both in serum and pFF or whether there is a particular beneficial influence from the follicular fluid itself. In general, it appears that the use of pFF is superior over serum as a supplement for in vitro maturation (IVM) of pig oocytes [10,11–14]. The most recent report suggested that IVM of porcine oocytes in NCSU-37 supplemented with FCS reduced the ability of the oocytes to mature, but the matured oocytes had the same ability to develop to term after IVF and embryo transfer compared to oocytes matured in pFF. However, different comparative studies concerning the supplementation of either pFF or serum resulted in inconsistent outcomes. This may be due to the inherent variability in the composition of pFF or serum. In this regard, several investigations concentrated on the source of pFF. The effect of pFF on overall in vitro production of porcine embryos is greatly depending on the follicle diameter of which it is obtained [14–16]. Previous studies agreed that application of pFF of the larger antral follicles resulted in improvement of IVP of porcine embryos compared to the fluids obtained from the smaller ones. However, with respect to the serum source, studies comparing serum and pFF mainly applied FCS probably because of its commercial availability. In this context, it is important to notice that FCS is derived from another species (heterologous), which might be an additional reason for the currently observed superiority of pFF compared to serum. In order to know whether the beneficial substances are present in both serum and pFF or whether they are produced locally in the follicle, it is a prerequisite to investigate both serum and pFF from the same animal at the same time in a comparative study. The aim of the present study was to compare the efficacy of supplementation of pFF from preovulatory follicles to the maturation medium with supplementation of autologous serum, derived from the same sows. We looked at various parameters of oocyte maturation, such as cumulus expansion and nuclear maturation. Moreover, subsequent sperm penetration and polyspermy rates and blastocyst development were determined, to evaluate a possible influence on cytoplasmic maturation. 2. Materials and methods 2.1. Materials All chemicals used in this study were purchased from Sigma–Aldrich (Bornem, Belgium) unless otherwise stated.

The basic medium used for the collection and washing of cumulus oocyte complexes (COCs) was a modified HEPES-buffered Tyrode balanced salt solution consisting of 3.1 mM KCl, 114 mM NaCl, 2 mM NaHCO3, 0.3 mM NaH2PO4, 2.1 mM CaCl2, 0.4 mM MgCl2, 10 mM sodium lactate, 0.2 mM sodium pyruvate, 10 mg/ ml gentamycin sulfate, 10 mM HEPES and 3 mg/ml BSA (HEPES-TM). The oocyte maturation medium was the BSA-free ‘North Carolina State University’ 23 (NCSU23) [17] supplemented with 0.57 mM cysteine, 10 ng/ml epidermal growth factor, 10 IU/ml equine Chorionic Gonadotropin (eCG) (Folligon1, Intervet, The Netherlands) and 10 IU/ml human Chorionic Gonadotropin (hCG) (Chorulon1, Intervet, The Netherlands). The basic medium used for IVF was designated as modified Tris-buffered medium (mTBM) and consisted of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl22H2O, 20 mM Tris (Trizma Base), 11 mM D-glucose and 5 mM sodium pyruvate and supplemented with 2 mM caffeine and 0.2% BSA [18]. The embryo culture medium was NCSU23 with 0.4% BSA (fraction V). 2.2. Preparation of pFF and serum Adult sows (n = 4) (Rattlerow-Seghers, Baasrode, Belgium) received 1500 IU eCG the day after weaning and 1500 IU hCG another 72 h later. After slaughter of the sows about 22 h after hCG administration, ovaries and blood samples of each sow were collected and transported to the laboratory within half an hour. Ovaries were transported in physiological saline at 37 8C while blood samples were transported at room temperature. Porcine follicular fluid was collected from follicles more than 6 mm in diameter, by means of an 18-G needle, which was attached to a disposable syringe. In the laboratory, pFF aspirates and blood samples were centrifuged (100  g, 10 min), the supernatant was aspirated, filtered (0.22 mm), snapfrozen and stored at 80 8C until use. 2.3. Oocyte collection and in vitro maturation Ovaries of prepubertal gilts were collected at a local slaughterhouse and transported to the laboratory in physiological saline at 37 8C. Oocytes were aspirated from non-atretic follicles (3–6 mm diameter). Oocytes with a homogeneous cytoplasm and a complete, dense cumulus were selected and washed. Groups of 80–120 oocytes were cultured into 500 ml maturation medium for the first 22 h (39 8C, 5% CO2 in air). Afterwards, oocytes were transferred to hormone-free maturation medium for another 22 h. During 44 h of IVM,

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maturation medium was supplemented with either10% serum, 10% pFF or 0.1% polyvinylalcohol (PVA). After IVM, oocytes were stripped of the cumulus by vortexing with 0.1% (w/v) hyaluronidase in HEPESTM. Zona pellucida (ZP)-free oocytes were prepared by incubating the oocytes briefly in a 0.1% pronase in PBS at 37 8C.

in the samples was calculated from a standard curve (0.03–0.5 nmol) and a blank sample. Glutathione content per oocyte was calculated by dividing the total content per sample by the number of oocytes present in the sample.

2.4. In vitro fertilization and in vitro culture of mature oocytes

After maturation or fertilization, groups of 5–10 oocytes/presumed zygotes were vortexed in 0.1% (w/v) hyaluronidase in HEPES-TM. After washing the oocytes/presumed zygotes in PBS, they were transferred to a 0.1% (w/v) pronase solution in PBS [21]. Zona pellucidae were continuously observed for dissolution under a stereomicroscope equipped with a warm plate at 37 8C. The dissolution time of the ZP of each oocyte was assessed at magnification 80 as the time interval between the first contact of the oocytes/ zygotes with the pronase solution and the first visible distortion of the ZP.

After IVM, cumulus-intact, cumulus-free or zonafree oocytes were washed three times in mTBM and transferred to droplets of 50 ml mTBM covered with mineral oil. Frozen, ejaculated sperm was thawed and processed through a 45%:90% Percoll gradient and centrifuged (700  g, 30 min). The resulting pellet was washed in mTBM by centrifuging (100  g, 10 min) and resuspended in mTBM. Sperm concentration was determined by ‘Computer assisted sperm analysis’ (CASA) and spermatozoa were added (50 ml) to the droplets containing the oocytes giving a final oocyte: progressive motile spermatozoa ratio of 1:3000 for cumulus-intact and cumulus-free oocytes and 1:300 for zona-free oocytes [19]. After a co-incubation period of 6 h, oocytes were vortexed in HEPES-TM to remove loosely bound spermatozoa, washed another three times in culture medium and cultured for 20 h or 7 days in droplets of 50 ml NCSU23 with 0.4% BSA. 2.5. Assay of GSH content The GSH content in the oocytes was determined as by Anderson [20] with some modifications. Oocytes were washed three times in an assay buffer (0.2 M sodium phosphate buffer containing 10 mM EDTA, pH 7.2) and groups of 20–30 oocytes in 5 ml of assay buffer were transferred to 1.5 ml microfuge tubes, to which 5 ml of 1.25 M H3PO4 was added. Samples were stored at 80 8C until assayed. The intracellular content of GSH was determined using the 5,50 -dithiobis(2nitrobenzoic acid)-glutathione disulfide (DTNBGSSG) reductase assay. A 700 ml of 0.29 mg/ml NADPH in assay buffer, 100 ml of 0.75 mM DTNB, 194 ml of water were added to the sample and mixed. Six ml of a 400 IU/ml glutathione reductase solution was added to start the formation of 5-thio-2-nitrobenzoic acid, which has an absorption peak at 412 nm. This was followed with a Beckman DU-600 spectrophotometer every 20 s for 2 min. The GSH content

2.6. Assessment of zona pellucida solubility

2.7. Assessment of oocyte maturation, fertilization and embryonic cell number Cumulus expansion was evaluated under a stereomicroscope (magnification 50) 22 h and 44 h after the start of maturation culture. Denuded oocytes, zygotes and embryos were fixed with 4% of paraformaldehyde in PBS, and subsequently stained with 10 mg/ml bisbenzamide (Hoechst 33342; Molecular Probes, Leiden, The Netherlands) in PBS (10 min). Nuclear DNA was visualized under a Leica DMR fluorescence microscope (Van Hopplynus NV, Brussels, Belgium). Oocytes with a metaphase plate and a polar body were classified as being in the MII stage. The presence of two pronuclei or cleaved embryos with two normal blastomeres was indicative for normal fertilization. Zygotes with more than two pronuclei or more than one decondensed sperm head were considered as polyspermic, whereas oocytes without penetrated sperm heads were considered as not fertilized. The total cell number of blastocysts was determined after 7 days of embryo culture. 2.8. Experimental design 2.8.1. Experiment 1: the effect of serum/pFF on nuclear maturation and fertilization of cumulusintact oocytes Cumulus oocyte complexes were cultured for 44 h in the presence of 10% pFF or 10% serum of three sows. Oocytes cultured in 0.1% PVA served as negative

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control. After 22 h and 44 h IVM, cumulus expansion was evaluated and a portion of the oocytes was used (n = 392) to assess nuclear maturation. The remaining cumulus-intact oocytes (n = 596) were fertilized and cultured for another 20 h to evaluate the fertilization state. 2.8.2. Experiment 2: the effect of serum/pFF on oocyte investments and on cytoplasmic maturation and polyspermic fertilization Based on the results of the previous experiment, a beneficial effect of pFF on cumulus expansion and monospermic penetration was detected. Hence, the possible role of oocyte investments on this outcome was investigated. After IVM of 44 h in either 10% serum or 10% pFF of one sow, some of the oocytes were denuded for assessment of ZP solubility (n = 296) and oocyte glutathione content (n = 149). The remaining oocytes were kept cumulus-intact (n = 343) or treated to obtain cumulus (n = 376) or zona-free oocytes (n = 177), which were fertilized afterwards. After gamete coincubation, a part of cumulus-intact presumed zygotes was used for ZP solubility (n = 289) determination whereas the other part was cultured in vitro for 20 h to analyze the fertilization status. All remaining cumulusand zona-free oocytes were used for determination of the fertilization status. 2.8.3. Experiment 3: the effect of serum/pFF on embryonic development Cumulus oocyte complexes were matured in 10% serum or 10% pFF of one sow. Cumulus-intact oocytes (n = 896) were fertilized and cultured as described above. After 7 days of embryo culture, blastocyst rates and blastocyst (n = 51) cell numbers were analyzed. 2.9. Statistical analysis The data concerning fertilization, blastocyst rates and nuclear maturation were analyzed using logistic regression analysis including the effect of replicate. Treatment was included as fixed factor and the experimental outcome (0–1) as dependent variable. For analysis of the data concerning embryonic cell number, oocyte glutathione content and ZP digestion time, analysis of variance including the effect of replicate was used. At least three replicates were conducted for each experiment. P-values less than 0.05 were considered significant (two-sided test). Statistical analysis was performed using SPSS 15.00.

3. Results 3.1. Experiment 1: the effect of serum/pFF on nuclear maturation and fertilization of cumulusintact oocytes For all sows observed, COCs matured in NCSU23 supplemented with pFF showed an increased cumulus expansion compared to those matured in serum (Fig. 1). This difference in cumulus expansion could not be extended to a significant difference on the overall nuclear maturation of the oocytes. In fact, the percentage of oocytes reaching the metaphase II stage was identical for both groups of maturation, with no differences between sows (86.0% versus 85.5% for serum versus pFF). In contrast, significantly fewer (P < 0.05) oocytes (41.0%) from the negative control group could be considered as fully mature. With regard to IVF, penetration rates were significantly lower when oocytes were matured in NCSU23 supplemented with pFF compared to serum (33.7% versus 47.4%) (P < 0.05) (Fig. 2). Of these penetrated oocytes, more monospermic zygotes were observed after maturation in pFF (78.3% versus 46.7%). In the negative control group, 16.7% of the oocytes were penetrated after the co-incubation period, 22.2% of them was penetrated by more than one spermatozoon. 3.2. Experiment 2: the effect of serum/pFF on oocyte investments and on cytoplasmic maturation and polyspermic fertilization After maturation of oocytes in the presence of pFF, a tendency for decreased zona solubility was observed, although the difference with oocytes matured in serum was borderline significant (P = 0.05) (81.2 s versus 77.4 s, respectively). After fertilization however, ZPs of presumed zygotes obtained from oocytes matured in pFF, were significantly more resistant to pronase compared to ZPs from presumed zygotes obtained from oocytes matured in serum (P < 0.05) (95.1 s versus 77.4 s) (Table 1). Moreover, a significantly higher GSH content/oocyte was found after maturation of oocytes in pFF (6.7 rmol/oocyte) versus serum (5.1 rmol/oocyte) (P < 0.05). After IVF of the cumulus-intact oocytes, the results of experiment 1 were confirmed since significantly higher rates of penetration (76.1% versus 62.2%) were detected after IVM in serum versus pFF (P < 0.05). Again, the higher penetration rates were due to an increase in polyspermy of oocytes matured in serum (66.1% versus 55.4%) (Fig. 3). However, rates of sperm penetration and polyspermic

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Fig. 1. Cumulus expansion after 22 h (A, C, E) and 44 h (B, D, F) IVM in NCSU23 supplemented with 0.1% PVA (A and B), 10% serum (C and D) or 10% pFF (E and F) versus immature compact COCs (G). After 22 h IVM, serum and pFF as supplements of the maturation medium were able to induce cumulus expansion, whereas no signs of cumulus expansion were observed for oocytes matured in 0.1% PVA. Cumulus cells were most expanded after maturation in 10% pFF (E). After 44 h IVM in 10% pFF, almost all oocytes were embedded in a cloud of expanded cumulus cells kept together in a viscoelastic matrix (F). However, after 44 h IVM in serum, the expanded cumulus matrices broke loose for most of the oocytes and the COCs sticked to the bottom of the multidish (D). No cumulus expansion was observed for COCs matured in 0.1% PVA (B). Bars = 100 mm.

fertilization in cumulus-free and zona-free oocytes were not affected (P > 0.05) by the presence of serum or pFF in the maturation medium. After IVF of cumulus-free oocytes, 84.9% versus 84.8% of the oocytes were

penetrated after IVM in serum and pFF, respectively, a similar number (79.6% versus 76.5%) of these oocytes was penetrated by multiple spermatozoa. For both groups of maturation, cumulus-free oocytes showed

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Fig. 2. Effect of supplementation of the maturation medium with either serum (S) or follicular fluid (pFF), obtained from three different sows, on fertilization of porcine cumulus-intact oocytes (S1: sow1; S2: sow2; S3: sow3). Height of the bars represents the mean  S.E.M. of normal fertilization (black) and polyspermic fertilization (grey) of four replicates. The overall height of the bars represents the penetration rate (grey + black). Within each sow, statistical analysis was performed to evaluate differences in penetration rate, polyspermy and normal fertilization. Different letters represent significant differences between penetration (a,b), polyspermy (h,i) and normal fertilization (x,y) between groups of oocytes matured in either serum or follicular fluid from the same sow (P < 0.05).

more polyspermic fertilization compared to IVF of cumulus-intact oocytes. After IVF of zona-free oocytes, no differences in penetration rate and polyspermic fertilization were observed. For oocytes matured in serum and pFF, 51.3% and 54.6% were penetrated and 50.0% and 55.6% of them were polyspermic, respectively.

Fig. 3. Effect of supplementation of the maturation medium with either serum (S) or follicular fluid (pFF)) on IVF of porcine cumulusintact (CI) and cumulus-free (CF) oocytes. Height of the bars represents the mean  S.E.M. of normal fertilization (black), polyspermic fertilization (dark grey) and penetration (light grey) of four replicates. Statistical analysis was performed to evaluate differences in penetration rate, polyspermy and normal fertilization between four groups of oocytes (CI S, CI pFF, CF S, CF pFF). Different letters represent significant differences in normal fertilization (a,b), penetration rate (x–z) and polyspermic fertilization (h–j) (P < 0.05).

number of blastocysts obtained from oocytes matured in pFF tended (P = 0.08) to be higher (19.9) than the cell number of blastocysts in the serum group (15.0) (Table 2). 4. Discussion

After 7 days of in vitro culture, the percentages of blastocysts obtained from oocytes matured in serum and pFF were 8% and 11%, respectively. However, the cell

Our results showed that (1) cumulus expansion and GSH content of the mature oocytes were increased after IVM in a pFF versus serum supplemented medium (2) pFF decreased polyspermic fertilization, with a concurrent reduction in overall penetration rate of cumulusintact oocytes, but these effects disappeared after removal of the cumulus and/or ZPs.

Table 1 Effect of IVM medium supplementation on zona pellucida solubility of porcine oocytes after IVM and IVF

Table 2 Effect of the IVM medium on embryonic development

3.3. Experiment 3: the effect of serum versus pFF on embryonic development

ZP dissolution time in 0.1% pronase (s) (mean  S.E.M.)

Unfertilized Fertilized

10% serum

10% pFF

81.2  11.1a 77.4  12.6a

92.4  12.5a 94.9  19.9b

pFF: porcine follicular fluid; S.E.M.: standard error of the mean. a,b Values within the same row with different superscripts represent statistical differences 25–30 oocytes/zygotes per group per replicate.

Group

Serum pFF

Embryonic development

Embryonic cell number

n

Blastocyst (%)

n

Mean cell number (S.E.M.)

336 314

26 (8) 34 (11)

24 27

15.0  3.0 19.9  2.2

Data from three (embryonic cell number) or four replicates (embryonic development). pFF: follicular fluid; S.E.M.: standard error of the mean.

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The cumulus expansion was remarkably more pronounced after both 22 h and 44 h of culture of the COCs in 10% pFF versus 10% serum. In vivo, cumulus expansion is induced after the preovulatory LH surge [22]. First of all, the cumulus cells undergo a cytoskeletal rearrangement which is a prerequisite for the deposition of the cumulus matrix [23]. In turn, the cumulus cells start the production of hyaluronan (HA), which is the main component of cumulus expansion or mucification. Hyaluronic acid is a high molecular weight glycosaminoglycan (GAG) but it is not linked to a core protein. Hence, it needs several hyaluronanbinding proteins (HABPs) to form the HA-rich viscoelastic matrices in the extracellular space. For in vitro cumulus expansion, the use of serum or pFF as a transudate of serum, is required to retain HA in a stable complex, which suggests the presence of those HABPs in serum and pFF [24,25]. Several HABPs have been identified in the pFF and they can be grouped either as serum-derived or follicle-specific proteins. Inter-atrypsin inhibitor (IaI) is a complex set of molecules derived from the serum and it is critical for cross-linking HA strands and stabilizing the cumulus matrix [26]. The product of tumor necrosis factor-stimulated gene-6 (TSG-6) is a glycoprotein that catalyzes the complex formation between IaI and HA during cumulus expansion of human oocytes [27,28]. Pentraxin 3, another protein synthesized by the cumulus cells, is also localized in the expanded extracellular matrix. Although it is not able to bind HA, pentraxin 3 has been shown to be necessary for matrix stabilization of murine oocytes probably by binding IaI [29]. Whether these highly important roles of pentraxin 3 and TSG-6 are also true for cumulus expansion of porcine oocytes remains to be elucidated, just as the question whether one of those above-mentioned follicle-specific component is responsible for the difference in cumulus expansion observed in the present study. Daen et al. [30] suggested the existence of a factor in pFF with a molecular weight smaller than 6.5 kDa involved in cumulus expansion. This factor is heat stable and insensitive to repeated cycles of freezing and thawing. However, Yoshida [31] suggested that the factor responsible for cumulus expansion is more than 200 kDa. Since the composition of pFF reflects changes in the secretory processes of the granulosa and theca cells in the follicle, the source of pFF may affect IVM and cumulus expansion of porcine oocytes. It has been suggested before that some factor(s) in pFF from ovarian follicles at later stages of follicular development may play an important role during oocyte maturation in vitro [15]. Moreover, the process of oocyte maturation

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in vivo takes place in the preovulatory follicle suggesting a highly supportive role of the preovulatory follicle contents on the maturation events. For practical reasons, we decided to use superovulation and to slaughter the sows at a specific time post-hCG injection. In this way, all sows were at the same stage of follicular growth at slaughter and possible individual variation was reduced. The gonadotropin treatment may involve some alterations of the composition of the pFF, but we are not aware of any publications who have specifically addressed this particular issue. However, a comparative study of the composition of autologous serum and pFF obtained at different stages of follicular growth may help to further identify this pFF specific factor(s) and its mechanism of action. In pig oocytes, as in some other mammals, the expanded mucified matrix surrounds the oocyte after its entrance in the oviduct and is consequently expected to play a role in in vivo fertilization [32]. It remains an important question whether extrapolation to in vitro conditions is warranted [33]. Our results showed a lower rate of polyspermic fertilization after IVF of cumulus-intact oocytes, accompanied by a regular decrease in penetration for oocytes matured in pFF compared to serum. This effect on polyspermy disappeared after removal of the cumulus cells. Gil et al. [34] had to use a four times higher sperm concentration for cumulus-intact oocytes compared to denuded ones to achieve a comparable penetration rate between both groups of oocytes. This study confirms our results that cumulus cells are able to prevent some spermatozoa from reaching the oocyte. The highly hydrated and viscoelastic matrices in the extracellular space during cumulus expansion may act as general mechanical barriers to sperm penetration of the oocyte. Moreover, spermatozoa possess a hyaluronidase activity which is necessary to penetrate the HArich matrices. Tatemoto et al. [35] recently showed the blocking activity of a chondroitin sulfate derived oligosaccharide on the hyaluronidase activity of the spermatozoa. Proteoglycans found in pFF predominantly contain chondroitin sulfate which might prevent the breakdown of the hyaluronan-rich matrix and consequently the number of spermatozoa reaching the oocyte. Spermatozoa are characterized by a HA-binding ability positively correlated with their motility. It might be possible that only motile spermatozoa are selected by the expanded cumulus matrix and that these are able to bind the extracellular matrix of the oocyte they try to invade [36]. Intact cumulus cells are known to produce glutathione (GSH) during IVM and this GSH can subsequently be accumulated in the ooplasm of the

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oocytes [37]. Glutathione is a major free thiol with important biological functions like protection of the oocytes against oxidative stress [38,39]. The value of intracellular GSH might be correlated with developmental competence of porcine oocytes matured in vitro and GSH has been used several times to evaluate different culture conditions [38]. In the present study, GSH content was higher after maturation in pFF compared to serum. Buthionine sulfoximide (BSO), an irreversible inhibitor of the g-glutamylcysteine synthetase, decreases bovine cumulus cell expansion and also inhibits GSH production [40], suggesting cumulus cell expansion may be correlated with GSH synthesis. The higher rate of cumulus expansion found in our study compared to the degree of cumulus expansion reported by Furnus et al. [40] could be a reason for the accompanying higher oocyte glutathione content for oocytes matured in the presence of pFF. In vitro fertilization of denuded oocytes with different glutathione contents did not result in an accompanying change in polyspermic fertilization as was seen after IVF of cumulus-intact oocytes with different rates of cumulus expansion. Consequently, cumulus expansion probably does not act through GSH in the oocyte in preventing polyspermic fertilization. Furthermore, increased cytoplasmic GSH content of mature porcine oocytes has previously been shown to have no immediate effect upon polyspermic fertilization and penetration rate [41,42]. Nevertheless, next to the earlier suggested possible role of the cumulus expanded matrix in preventing penetration of the oocyte by multiple spermatozoa, we also found a significant increase in zona digestion time of fertilized oocytes matured in pFF. Zona hardening, as the main block to polyspermic fertilization of porcine oocytes, is characterized by an increased resistance of the ZP to proteolytic digestion [21,43]. Thus, our results suggest an improved zona hardening after IVF of oocytes matured in pFF. However, the time required for zona digestion for oocytes matured in pFF is still far beneath that of in vivo matured oocytes once they become exposed to spermatozoa. The time necessary to dissolve the ZP for in vivo matured pig oocytes collected directly from the preovulatory follicles is about 6 min, but can be extended to several hours after ovulation [44]. Moreover, after culturing pig oocytes in 30% oviductal fluid, there was an increase in the time necessary for ZP digestion and a decrease in polyspermic fertilization, which suggests the overall importance of the oviductal secretions to ensure the hardening of the ZP [21,45]. Even though ZP hardening is usually understood as a common event in mammals,

several authors were faced with the absence of ZP hardening during IVF of porcine oocytes. Coy et al. [46] proposed a delayed or impaired in vitro cortical reaction as a possible explanation for the absence of ZP hardening while Wang et al. [43] emphasized on the oviductal contact as a necessity for zona hardening. Before we can concur with one of the hypotheses mentioned above, further investigation should be performed. Nevertheless, the finding that polyspermic fertilization rates remained unchanged after fertilization of zona-free oocytes, matured in one of both in vitro conditions, suggests that under these in vitro circumstances, the membrane block is of little relevance. The suggested delayed or incomplete zona reaction and membrane block for in vitro conditions may be partly overcome by the presence of the cumulus cells during in vitro fertilization of porcine oocytes. In conclusion, both autologous serum and pFF support nuclear maturation, fertilization and embryonic development although an increased cumulus expansion was observed after IVM of COCs that were matured in pFF. These expanded cumulus matrices surrounding the oocytes presumably prevent polyspermic penetration during IVF in a mechanical way. Further investigation is needed to elucidate the biochemical and functional nature of the follicular fluid specific factors involved in this increased cumulus expansion. Nevertheless, also factors derived from serum need some future investigation since their beneficial effects on in vitro production of porcine embryos may not be underestimated. Acknowledgements This investigation was supported by a grant of ‘Bijzonder Onderzoeksfonds’ (BOF) of the Ghent University of Belgium. The authors owe thanks to Bart Buysse and Nadine Buys of Rattlerow-Seghers, Baasrode, Belgium for providing the sows in the preovulatory phase of the estrous cycle, and to Johanna Mestach, Sofie Vandenabeele and Isabel Lemahieu for their excellent technical assistance. References [1] Edwards RG. Follicular fluid. J Reprod Fertil 1974;37:189–219. [2] Abeydeera LR, Wang W, Prather RS, Day BN. Maturation in vitro of pig oocytes in protein-free culture media: fertilization and subsequent embryo development. Biol Reprod 1998;58: 1316–20. [3] Hong JY, Yong HY, Lee BC, Hwang WS, Lim JM, Lee ES. Effects of amino acids on maturation, fertilization and embryo development of pig follicular oocytes in two IVM media. Theriogenology 2004;62:1473–82.

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