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Regular Article |
a Centre de Recherche en Biologie de la Reproduction, Department of Animal Science, Laval University, Ste-Foy, Quebec, Canada G1K 7P4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, fertilization, follicular development, in vitro fertilization, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When immature bovine oocytes are released from their follicles and are cultured in standard maturation medium, they resume the first meiotic division [3]. Although it seems difficult to influence oocytes during their maturational period to modulate their response for subsequent development, the alteration of basic maturation conditions can significantly affect oocyte competence as reflected by the morula and blastocyst yield after in vitro fertilization (IVF) [4].
During IVM, oocytes undergo a series of cytoplasmic changes before the resumption of nuclear maturation, leading to variable competence of the resulting embryo [5]. In addition, the synthesis and storage of certain forms of mRNA and protein during IVM and early embryonic development are thought to be necessary for further development [6, 7].
The limited developmental competence of bovine oocytes after IVM can be used to understand the factors involved in the acquisition of such ability. For this reason and to understand the requirements for development of immature oocytes through IVM, all products with undefined components should be eliminated from culture conditions. Although serum or BSA are typically added to the medium as a protein supplement to improve culture efficiency [8], different lots of this protein can produce highly variable effects during the period of culture in hamsters, ranging from highly stimulatory to highly inhibitory [9].
To elucidate the roles of protein supplements on oocyte maturation, various proteins including BSA-V, purified BSA, essentially fatty acid-free BSA (BSA-FAF), chicken egg albumin, and fetal calf serum (FCS) were tested for their effects on oocyte maturation and subsequent embryonic development after IVF. The replacement of these proteins with synthetic macromolecules such as polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA) was also evaluated in defined maturation medium. Medium with no supplementation was also evaluated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bovine ovaries were transported from the slaughterhouse to the laboratory in a 0.9% NaCl aqueous solution containing 100 000 IU/L penicillin, 100 mg/L streptomycin, and 250 µg/L amphotericin B (Sigma-Aldrich, Oakville, ON, Canada). COCs were aspirated from 2- to 6-mm follicles using an 18-gauge needle attached to a 10-ml syringe. COCs with a homogenous cytoplasm and multiple compact layers of cumulus cells were selected and washed three times in synthetic oviduct fluid medium (SOF) supplemented with modified Eagle medium (MEM) nonessential amino acids (Gibco BRL, Burlington, ON, Canada), MEM essential amino acids (Gibco), 0.4 mM pyruvic acid, 1 mM glutamine (Sigma-Aldrich), and 50 µg/ml gentamicin (Sigma-Aldrich).
In Vitro Maturation
COCs were incubated in groups of 10. Each 50-µl droplet of maturation medium consisted of modified SOF supplemented with MEM nonessential amino acids (Gibco), MEM essential amino acids (Gibco), 1.5 mM glucose (Sigma-Aldrich), and 1 mM glutamine (Sigma-Aldrich). The droplets were covered with mineral oil (Sigma-Aldrich), preincubated under the maturation conditions for a minimum of 3 h (38.5°C, 5% CO2 in air with 100% humidity), and then incubated for 24 h after oocytes were added.
In Vitro Fertilization
IVF took place in droplets composed of modified Tyrode lactate (TL) medium [10] supplemented with 0.6% BSA-FAF (Sigma-Aldrich), 0.2 mM pyruvic acid, 2 µg/ml heparin (Sigma-Aldrich), and 50 µg/ml gentamicin containing five oocytes. COCs were previously washed twice for 5 min in Hepes-buffered TL medium (TLH). Following the transfer of oocytes, 2 µl of PHE (2 mM penicillamine, 1 mM hypotaurine, 250 mM epinephrine; Sigma-Aldrich) was added to each droplet. All experiments were carried out using frozen semen from the same bull (Centre d'Insémination Artificielle du Québec, St-Hyacinthe, PQ, Canada). Spermatozoa were thawed in a 35°C water bath for 1 min and then washed in a discontinous Percoll gradient prepared by adding 2 ml of 90% Percoll under 2 ml of 45% Percoll in a 15-ml centrifuge tube (Falcon, Franklin Lakes, NJ). The semen samples were added onto the Percoll gradient and centrifuged at 700 x g for 30 min at 26°C. The pellet was removed and resuspended in 1 ml of modified TL medium (TALP) and centrifuged at 250 x g for 5 min at 26°C. After removal of the supernatant, spermatozoa were resuspended in IVF medium and counted in a hemocytometer chamber, and 2 µl of sperm suspension (final concentration = 106 cells/ml) was added into each droplet (final volume 52 µl). Incubation was carried out at 38.5°C in 5% CO2 in air with saturated humidity for 15–18 h.
In Vitro Culture
In all experiments, embryo culture took place in modified SOF under mineral oil in a humidified atmosphere of 5% CO2 and 7% O2 at 38.5°C. Between 15 and 18 h after insemination, presumptive zygotes were denuded of surrounding cumulus cells by repeated pipetting in PBS and subsequently washed three times in PBS before being transferred to the culture droplets (50 µl) in groups of 20–30 embryos. Cleavage was assessed after 72 h of culture, and the number of embryos developing to the morula and blastocyst stages was assessed on Day 8.
To prevent toxic accumulation of ammonium as a result of amino acid degradation, SOF medium was replaced every 72 h. In this study, we used a two-culture system. The first system (SOFC1) medium contained 0.8% BSA-V (Sigma-Aldrich), MEM nonessential amino acids, 1 mM glutamine, 1.5 mM glucose (Sigma-Aldrich), and 10 µM EDTA (Sigma-Aldrich) for the first 72 h. Then, the medium was replaced by the second system (SOFC2) containing 0.8% BSA-V, MEM nonessential amino acids, MEM essential amino acids, 1.5 mM glucose, and 1 mM glutamine for the remaining 96 h of culture.
Experimental Design
Experiment 1 In each of three replicates, the effects on oocytes of 1, 8, or 20 mg/ml BSA-V (lot 89H1270, Sigma-Aldrich) added to SOF during IVM were compared with the effects of control medium alone (BSA-free IVM medium).
Experiment 2 To investigate the effect of different sources of protein on bovine oocyte maturation and subsequent embryo development, COCs were cultured in SOF in the presence of BSA-V (lot 89H1270, Sigma-Aldrich), purified BSA (lot 79H7614, Sigma-Aldrich), BSA-FAF (lot 16H9310, Sigma-Aldrich), chicken egg albumin (lot 60K0844, Sigma-Aldrich), or FCS (lot 10027-1; MEDICORP, Montréal, PQ, Canada). COCs cultured without protein supplementation served as controls.
Experiment 3 To investigate the effects of synthetic macromolecules added during IVM of bovine oocytes, COCs were cultured in SOF in the presence of BSA-V, PVP-40 (Mr 40 000), PVP-360 (Mr 360 000), PVA, or FCS.
Experiment 4 To study the effect of BSA-V on nuclear maturation, COCs were cultured in the presence of 8 mg/ml BSA-V, 10% FCS, and 8 mg/ml PVP. The percentage of oocytes in germinal vesicle (GV) and metaphase II (MII) stages were determinted after 6 h and 18 h, respectively, of incubation. Oocytes cultured without protein supplementation served as controls.
Experiment 5 The aim of this experiment was to determine the effect of recombinant human FSH (r-hFSH) on cumulus expansion and nuclear maturation under defined conditions in medium containing BSA-V or PVP-40. Cumulus expansion was evaluated under a stereomicroscope at the end of the incubation period (18 h) prior to the vortex treatment (-, no expansion; +, intermediate or partial expansion; ++, complete expansion).
Oocyte Fixation
COCs were cultured in maturation medium supplemented with different proteins (BSA-V, FCS, or PVP) for the first 6 and 18 h. COCs were transferred into small centrifuge tubes containing 500 µl of TLH and vortex-agitated for 4–5 min to remove the cumulus cells. The denuded oocytes were recovered under a stereomicroscope and transferred onto a glass slide in a small drop. A vaseline:paraffin (3:1) mixture was used to maintain the coverslip in contact with the oocytes. The slides were then immersed in ethanol:acetic acid (3:1) for a minimum of 24 h before staining with 1% aceto-orcein and were examined for nuclear morphology with a phase contrast microscope at 400x magnification [11].
Statistical Analysis
Data from three replicates were expressed as mean ± SEM. The data were analyzed by ANOVA using the STATVIEW program. All percentage data were subjected to arcsine transformation before analysis. The Fisher least significant difference test was used to test the differences between treatments. A P value of <0.05 indicated a significance difference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In experiment 2, oocytes were matured in protein-free maturation medium or in the presence of different sources of albumin: BSA-V (8 mg/ml), purified BSA (8 mg/ml), BSA-FAF (8 mg/ml), chicken egg albumin (8 mg/ml), or FCS (10%). There were no significant differences in cleavage rate among treatments (Table 2). The presence of BSA-V and FCS significantly decreased the percentage of morulae and blastocysts (P < 0.05). However, SOF without protein supplementation significantly improved the percentage of embryos produced. When BSA-V was replaced with purified BSA, BSA-FAF, or chicken egg albumin as a protein supplement during IVM of bovine oocytes, the percentage of embryos developing after IVF improved.
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In experiment 3, after IVM of bovine oocytes in the presence of synthetic macromolecules (PVP or PVA) or protein (BSA or FCS), there were no significant differences in cleavage rate among treatments (Table 3). However, the development of embryos to the morula and blastocyst stages was improved by the addition of PVP-40 to the medium used for IVM as compared with addition of BSA, PVP-360, PVA, or FCS. SOF alone also supported oocyte maturation, as indicated by the morula and blastocyst yield.
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In experiment 4, addition of BSA-V to the maturation medium retarded the process of GV breakdown (GVBD) (Table 4). At 6 h, the percentage of oocytes in the GV stage in the BSA-V- or PVP-360-treated groups was significantly higher (P < 0.0001) than that in the control, and FCS-treated, and PVP-40-treated groups. The number of oocytes in the MII stage after 18 h of culture was significantly lower (P < 0.05) in the BSA-V group than in the control, FCS-treated, and PVP-40-treated groups.
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In experiment 5, the effect of r-hFSH on the number of oocytes in the GV and MII stages after 6 and 18 h of culture in the presence of BSA-V or PVP-40 is presented in Table 5. At 6 h, the percentage of oocytes in the GV stage was significantly higher (P < 0.05) in the BSA + r-hFSH-treated group than in the PVP-40 + r-hFSH-treated group. The proportion of oocytes in the MII stage at 18 h of maturation in the presence of BSA + r-hFSH was significantly lower (P < 0.05) than that in the presence of PVP-40 + r-hFSH. Cumulus expansion was maximal (complete expansion) in the presence of r-hFSH with BSA and intermediate (partial expansion) in the presence of PVP-40 + r-hFSH.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The negative effect of BSA-V seen in this study could be physiological (changing a function) or toxic. To discriminate between these two possibilities, COCs were matured in the presence of different albumin sources. When BSA-V was replaced with purified BSA, BSA-FAF, or chicken egg albumin, the embryo development rate was improved (Table 2). Therefore, we cannot exclude the possibility that the presence of BSA-V alone had a toxic effect and delayed the process of oocyte maturation. A low molecular weight contaminant in BSA-V preparations could act negatively when added to IVM medium. Different preparations of BSA have had various effects on development, ranging from highly stimulatory to highly inhibitory, in the hamster [9].
Another approach for studying the effect of BSA is to examine maturation kinetics. In vivo, cumulus cell expansion normally follows GVBD by a few hours [12]. During IVM, the process of cumulus cell expansion has an impact on fertilization rate and subsequent embryonic development up to the blastocyst stage [13, 14]. In the present study, no cumulus expansion was observed when oocytes were cultured in SOF with PVP or in SOF without BSA compared with culture in SOF with serum (FCS). In a previous study, IVM of bovine oocytes in the presence of low concentrations of invasive adenylate cyclase resulted in no cumulus expansion, but the blastocyst rate was significantly greater than that for oocytes matured in the presence of serum and gonadotropins, where cumulus expansion was high [15]. Thus, during IVM of bovine oocytes, the presence of cumulus cells might be necessary to express the competence of oocytes, but cumulus expansion is not required to improve competence; at least there is no direct relationship between cumulus expansion and further cytoplasmic maturation.
To verify the possible effect of BSA on maturation kinetics, COCs were cultured in maturation medium supplemented with BSA-V, PVP, or FCS for the first 6 or 18 h, and then nuclear morphology was examined. These two time points (6 h for GVBD and 18 h for the beginning of metaphase II stage) were selected to evaluate the ability of these proteins to stimulate or prevent oocytes from maturing at a normal rate [16]. Our results indicate that bovine oocytes are sensitive to BSA-V as a protein supplement; this supplement retarded GVBD and MII in a high proportion of the oocytes after 6 h and 18 h, respectively, of culture (Table 4). To verify that these results were not the consequence of a contaminated lot of BSA, COCs were cultured in maturation medium supplemented with a different lot of BSA-V (lot 98H1446). Addition of this BSA-V to the maturation medium also retarded nuclear maturation in bovine oocytes, as reflected by the higher proportion of GV and lower proportion of MII oocytes after 6 h and 18 h, respectively, of culture (data not shown). Maturation of bovine oocytes in SOF alone (without protein supplements) or in SOF with PVP-40 did not slow nuclear maturation and enhanced subsequent embryonic development. Addition of PVP-360 to the maturation medium also retarded nuclear maturation in bovine oocytes. The fact that more oocytes remained at the GV stage at 6 h (Table 4) may indicate an inhibitory effect of this supplement (PVP-360) on the meiosis induction mechanism.
Data from our laboratory have shown that addition of r-hFSH during IVM of bovine oocytes in the presence of BSA-V or PVP-40 under defined conditions significantly enhances oocyte developmental competence, as reflected by the morula and blastocyst yield obtained after IVF (unpublished results). The initial inhibitory action of FSH on oocyte maturation might be the result of elevated cAMP levels, and this gonadotropin may become stimulatory when the amount of cAMP accumulated generates a positive signal in the cumulus cells [17]. We presently have no information about the mechanism by which FSH affects nuclear maturation in the presence of BSA-V compared with PVP-40 in bovine oocytes under defined conditions during IVM.
Our results clearly demonstrated that IVM of bovine oocytes in the presence of BSA retards nuclear maturation. The lower proportion of GV oocytes after 6 h of culture in the presence of r-hFSH + PVP-40 compared with r-hFSH + BSA-V and the higher proportion of MII oocytes in the presence of r-hFSH + PVP-40 compared with r-hFSH + BSA-V may indicate a differential action of FSH on the oocyte or the surrounding cumulus cells. Recombinant hFSH could be inhibitory in the presence of BSA-V because of a greater response to adenylate cyclase and higher levels of cAMP, whereas r-hFSH in the presence of PVP-40 may be stimulatory because lower levels of cAMP could augment maturation but still be below the threshold required to cause inhibition. The higher degree of cumulus expansion by r-hFSH + BSA-V (complete expansion) compared with r-hFSH + PVP-40 (partial expansion) supports this hypothesis. These studies are the first to show that the effect of FSH on nuclear maturation and cumulus expansion is dependent on substrates present in IVM medium (Table 5).
In the present study, the ability of oocytes to develop to the blastocyst stage was not improved after maturation in the presence of serum, which was unexpected. This lack of improvement was reflected by the lower morula and blastocyst yield after IVF compared with culture in SOF alone despite the fact that most of the oocytes reached GVBD and MII after 6 h and 18 h, respectively, as in SOF alone. Our finding is in contrast to the previous result obtained by Leibfried-Rutledge et al. [8]; who demonstrated that FCS was a superior protein supplement compared with the often used 0.8% BSA for IVM of hamster and cow oocytes using modified Tyrode solution. As indicated by Lonergan et al. [18], the presence of serum during IVM of bovine oocytes using TCM-199 significantly enhanced oocyte maturation and subsequent embryonic development when compared with the use of SOF supplemented with serum. These results may suggest that the effect of FCS as a protein supplement during IVM of bovine oocytes depends on the culture medium used.
The role of albumin during culture is still unclear. There are several known functions of albumin in culture medium. Albumin chelates heavy metals and provides some pH buffering, which can also be achieved by inclusion of amino acids in culture medium [19]. The surfactant property of albumin prevents adhesion of cells to glass and plastic surfaces, but this effect can be achieved by synthetic polymers such as PVA [20–22]. Albumin also can act as a scavenger of reactive oxygen species [23].
In our study, replacement of PVP-40 with PVA decreased embryo development, particularly at the morula and blastocyst stages. This finding confirms the results obtained by Pinyopummintr and Bavister [23], who demonstrated that embryo development to the blastocyst stage was reduced by replacing BSA or serum with PVA.
Saeki et al. [24] examined the effect of PVP on the maturation of bovine oocytes and their subsequent development in vitro. These authors found that the presence of PVP during maturation in the absence of hormones (FSH, LH, and estradiol) yielded no blastocysts. In another study, however, the beneficial effect of PVP during maturation on oocytes was reflected by the morula and blastocyst yield obtained in the presence of various concentrations of gonadotropins and/or granulosa cells [25, 26].
In our study, when serum and BSA-V were replaced by synthetic macromolecules such as PVP-40 but not PVP-360 more embryos developed to the morula and blastocyst stages compared with IVM medium supplemented with serum or BSA (Table 3). These results indicate that bovine oocytes can be matured in vitro in serum- and BSA-free medium or in medium lacking protein, with no negative effect on further embryonic development. However, protein supplementation is important in fertilization and further development in bovine embryos.
The presence of protein seems to play a significant role for accelerating formation of pronuclei [27]. Wrenzycki et al. [28] reported recently that during in vitro culture of bovine embryos the protein source (serum, BSA, or PVA) had a weak but visible effect on the amounts of specific transcripts when compared with the basic culture system.
In the present study, we showed that protein supplements during IVM can have profound effects both on the rate of development and on the overall efficiency of development as shown by the morula and blastocyst yields. We successfully cultured bovine oocytes in maturation medium without protein supplement and in the presence of a synthetic macromolecule (PVP-40). These systems provide a favorable environment for studying the effect of defined components during IVM.
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FOOTNOTES |
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1 This work was supported by the Natural Sciences and Engineering Research Council of Canada. 
2 Correspondence. FAX: 418 656 3766; marc-andre.sirard{at}crbr.ulaval.ca
Accepted: October 31, 2001.
Received: September 18, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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