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RESEARCH |
Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, Murcia 30071, Spain
Correspondence should be addressed to P Coy; Email: pcoy{at}um.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The resumption of meiosis is regulated by activation of the metaphase promoting factor (MPF; Wu et al. 1996, Motlik et al. 1998, Sirard et al. 1998). MPF is a cell division kinase (cdk) whose activation involves the formation of a complex between p34cdc2 kinase (cdk1) and cyclin B. Once formed, this complex should be activated by dephosporylation of the threonine 14 and tyrosine 15 residues of the p34 subunit. The most common approach to block MPF activation and, consequently, the resumption of meiosis, has been the employment of cdk inhibitors. Roscovitine actively competes for the ATP binding sites in the cdk1 subunit of the MPF, and was first used to prevent germinal vesicle breakdown (GVBD) in cattle and pig oocytes (Mermillod et al. 2000, Krischek & Meinecke, 2001, Marchal et al. 2001, Ponderato et al. 2001, 2002). Specifically, the inhibitory effect of roscovitine on the cell cycle was discovered by Meijer (reviewed by Meijer & Raymond 2003). The results seem to demonstrate that it is more effective and produces less detrimental effects than other inhibitors such as cycloheximide (Faerge et al. 2001), butyrolactone I (Kitagawa et al. 1993, Meijer & Kim 1997, Kubelka et al. 2000, Fair et al. 2002) or 6-DMAP (Lonergan et al. 1997, Avery et al. 1998, Liu et al. 1998, Dode & Adona 2001).
Despite the numerous papers published in recent years, and even though studies in cattle have been more extensive than in pigs, no references are available showing the influence of pre-culture with roscovitine and further IVM on important parameters such as the nuclear stage during the maturation period, the intracellular glutathione (GSH) content of pre-cultured oocytes or male pronuclear formation after IVF of these gametes. Moreover, the birth of offspring has yet to be reported, and therefore also unreported is the competence to develop to term of embryos produced in vitro under the two-step IVM conditions. As a consequence, the overall objective of this experimental work was to assess the influence of roscovitine on the nuclear and cytoplasmic maturation of oocytes entering a two-step IVM system and to demonstrate its effectiveness to generate embryos with full-term developmental ability.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The basic medium used for IVF was essentially the same as that used by Rath et al.(1999). This medium, designated as TALP medium, consists of: 114.06 mmol/l NaCl, 3.2 mmol/l KCl, 8 mmol/l Ca-lactate·5H2O, 0.5 mmol/l MgCl2·6H2O, 0.35 mmol/l NaH2PO4, 25.07 mmol/l NaHCO3, 10 ml/l Na-lactate, 1.1 mmol/l Na-pyruvate, 5 mmol/l glucose, 2 mmol/l caffeine, 3 mg/ml BSA (A-9647), 1 mg/ml polyvinyl alcohol (PVA) and 0.17 mmol/l kanamycin sulfate.
The embryo culture medium was NCSU-23 containing: 0.4% BSA (A-8022), 75 µg/ml potassium penicillin G and 50 µg/ml streptomycin sulphate (Machaty et al. 1998).
Oocyte collection and in vitro maturation
Within 30 min of slaughter, ovaries from prepubertal gilts were transported to the laboratory in saline containing 100 µg/ml kanamycin sulfate at 38 °C, washed once in 0.04% cetrimide solution and twice in saline. Oocyte-cumulus cell complexes (COCs) were collected from antral follicles (3–6 mm diameter) by slicing, washed twice with Dulbecco’s PBS supplemented with 1 mg/ml PVA and twice more in maturation medium previously equilibrated for a minimum of 3 h at 38.5 °C under 5% CO2 in air. Only COCs with a complete and dense cumulus oophorus were used for the experiments (Coy et al. 2002). Groups of 50 COCs were cultured in 500 µl maturation medium for 22 h at 38.5 °C under 5% CO2 in air. After culture, oocytes were washed twice in fresh maturation medium without dibutyryl cAMP, eCG and hCG and cultured for an additional 20–22 h (Funahashi & Day 1993). This IVM system is commonly employed in pigs for several years and is based upon the results from Funahashi et al.(1997).
Brilliant cresyl blue test
Immediately after collection, COCs were washed in PBS and exposed to brilliant cresyl blue (BCB; 13 µmol/l in PBS) for 90 min at 38.5 °C in a humidified air atmosphere. BCB staining determines the activity of glucose-6-phosphate dehydrogenase, an enzyme synthesized in growing oocytes but with less activity in grown oocytes (Roca et al. 1998, Rodríguez-González et al. 2002). After exposure to BCB, they were washed three times in PBS and observed under a stereomicroscope at x 20 and classified into two groups, depending on their cytoplasm coloration: oocytes showing blue cytoplasm or grown oocytes (BCB+) and oocytes without blue coloration or growing oocytes (BCB–).
Hoechst staining
Oocytes were fixed for 15 min (2% glutaraldehyde in PBS), stained for 15 min (1% Hoechst 33342 in PBS) and finally washed in PBS containing 1 mg/ml polyvinylpyrro-lidone and mounted on glass slides. Oocytes were examined under an epifluorescence microscope at x 200 and x 400 magnification and designated as GV-0, GV-I GV-II, GV-III, GV-IV (including diakinesis and prometaphase I), metaphase I (Met I), anaphase I (Ana I) and Met II stages according to the morphological criteria for characterization of meiotic stages by Funahashi et al.(1997).
GSH assay
The intracellular content of GSH was measured as described previously (Funahashi et al. 1995). Briefly, COCs were denuded by pipetting in PBS and washed three times in a buffer solution. Five microliters of buffer containing 30 oocytes per replicate and group were transferred to a 2 ml microfuge tube and 5 µl of 1.25 mol/l phosphoric acid added. Samples were frozen immediately (–20 °C) and kept in the freezer until assayed. The GSH content in the oocytes was determined by the dithionitro-benzonic acid–glutathione disulphide (DTNB–GSSG) reductase recycling assay (Anderson 1985). Briefly, 700 µl of 0.33 mg/ml NADPH in 0.2 mol/l sodium phosphate buffer containing 10 mmol/l EDTA (stock buffer, pH 7.2), 100 µl of 6 mmol/l 5,5'-dithiobis-(2-nitrobenzoic acid) in the stock buffer, and 190 µl of water were added into the microfuge tube. Ten microliters of 250 iu/ml glutathione reductase were added with mixing to initiate the reaction. The formation of 5-thio-2-nitrobenzoic acid was detected photometrically by a change of absorption at 412 nm. The total GSH content is calculated according to a standard curve. The total amount of GSH calculated was divided by the number of oocytes in the sample to obtain the content per oocyte (pmol/oocyte).
In vitro fertilization
COCs cultured for a total of 44 h in maturation medium were stripped of cumulus mechanically by gentle aspiration with a pipette. Oocytes were washed three times with TALP medium and groups of 30–35 oocytes were transferred into each well of a four-well multi-dish containing 250 µl IVF medium previously equilibrated at 38.5 °C under 5% CO2. The sperm-rich fraction of semen from a mature, fertility-tested boar was collected by the gloved-hand method and immediately transported to the laboratory diluted 1:8 in Beltsville thawing solution. The semen samples were centrifuged (1200 g, 3 min) and the resultant sperm pellets were diluted in TALP medium at the desired concentration (Matás et al. 2003). The sperm suspension (250 µl) was added to each fertilization well to obtain a final concentration of 3000 cells/oocyte. At 5 h post-insemination (hpi), oocytes were washed twice with fresh NCSU-23 by gentle aspiration through a glass pipette and allowed to continue in culture at 38.5 °C under 5% CO2 until fixation or transfer.
At 18 hpi, a sample of oocytes was stained with Hoechst 33342 as described above and examined at x 400 magnification for evidence of sperm penetration and pronuclear formation under an epifluorescence microscope.
Embryo transfer
Multiparous (two pregnancies) crossbred sows with synchronized estrus cycles were used for surgical embryo transfer. Estrus was checked daily in the presence of a mature teaser boar. Occurrence of estrus was defined by the standing reflex in front of a boar (back-pressure test) and reddening and swelling of the vulva. Oviducts from animals which had showed heat 72 h before embryo transfer were exposed through mid-ventral incision under general anesthesia, as previously described (Coy et al. 1993). Over 100 potential embryos (22–24 hpi) were introduced in one oviduct by a Tom Cat Catheter (Kendall Co., Mansfield, MA, USA) connected to an insulin syringe containing PBS at 37 °C. The catheter was later observed under a stereomicroscope to check that all embryos had been transferred into the oviducts. The sows were kept under the usual farm conditions, and 25–28 days after transfer pregnancy diagnosis was carried out by ultrasonography (Echoscan T-100).
Experimental design
The experimental design is schematically represented in Fig. 1
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Experiment 1. Nuclear status after 22 h culture in roscovitine
This experiment was carried out to establish whether culture of oocytes in NCSU-37 medium without dibutyryl cAMP, eCG and hCG and with 50 µmol/l roscovitine for the first 22 h of maturation (ROS group, n = 152) kept the nuclear stage at the same level as found in oocytes just after recovery (Before culture group, n = 156). As control groups, oocytes cultured for 22 h in supplemented NCSU-37 medium were used under usual IVM conditions, which included dibutyryl cAMP, eCG and hCG (A group, n = 173) and oocytes cultured for 22 h in the same NCSU-37 medium without dibutyryl cAMP, eCG and hCG (B group, n = 164). Therefore, it could be seen whether the inhibitory effect of roscovitine on the nuclear progression was similar, or dissimilar, to that demonstrated by dibutyryl cAMP, which only blocks oocytes at stages before the GV-II stage (Funahashi et al. 1997); the nuclear stage of inhibited oocytes (ROS and A groups) at 22 h and of those under supposed spontaneous resumption of meiosis (B group) could also be compared.
After culture, oocyte nuclear status (GV-0 to Met II) was recorded in all groups. This experiment was performed in four replicates.
Experiment 2. Nuclear progression after IVM with a pre-maturation period in roscovitine
In order to assess the reversibility of the treatment with roscovitine, COCs were cultured for 44 h under permissive maturation conditions in each of three replicates with (ROS-IVM group, n = 153) or without (IVM group, n = 161) a previous culture period of 22 h in the presence of 50 µmol/l roscovitine. Nuclear stage was recorded by Hoechst staining.
Experiment 3. Oocyte GSH content after roscovitine treatment
This experiment was designed to examine the influence of roscovitine on the intracellular GSH content of oocytes just after collection, at 22 h of culture with or without roscovitine, and after maturation with or without a pre-culture in roscovitine. In seven replicates, 960 COCs were collected and allotted to five groups: (1) Before culture group (oocytes processed just after collection); (2) A group (oocytes cultured in supplemented NCSU-37 medium with dibutyryl cAMP, eCG and hCG); (3) ROS group (oocytes cultured for 22 h in supplemented NCSU-37 medium without dibutyryl cAMP, eCG and hCG and with 50 µmol/l roscovitine); (4) IVM group (oocytes cultured for 44 h under the described step-wise IVM system); (5) ROS-IVM group (oocytes cultured for 22 h as the ROS group and for a further 44 h as the IVM group). GSH content was measured as described above.
Experiment 4. IVF and full development of roscovitine-treated oocytes
Just after recovering, 631 oocytes (in four replicates) were cultured in the presence of 50 µmol/l roscovitine for 22 h. Following this period of time, oocytes were washed and allowed to mature for 44 h under permissive conditions (ROS-IVM group). Other 610 COCs were also collected and introduced in the IVM system at the same time as the ROS-IVM group. Matured oocytes from both groups were then fertilized and 18 hpi, samples of the potential embryos (n = 133 and n = 127 for ROS-IVM and IVM groups respectively) were processed to assess penetration and monospermy rates. The remaining cells (498 for ROS-IVM and 483 for ROS groups) were transferred into the oviduct of physiologically synchronized sows (one sow per group and replicate, eight sows in total). Pregnancies were assessed by ultrasonography 25–28 days after transfer.
Statistical analysis
Data are presented as means±S.E.M. and all percentages were modeled according to the binomial model of variables. The variables in all the experiments were analyzed by one-way ANOVA. These variables were as follows: experiments 1 and 2, the nuclear stage (GV-0 to Met II); experiment 3, the GSH content; experiment 4, the percentage of penetration, the number of sperm cells per penetrated oocyte, the male pronuclear formation and the putative embryos (putative embryos were defined as the percentage of monospermic oocytes with two pronuclei with respect to the total number of penetrated oocytes). When ANOVA revealed a significant effect, values were compared using Tukey’s test. P values < 0.05 were taken to denote statistical significance.
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Results |
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Nuclear status after 22 h in roscovitine
Roscovitine was effective in inhibiting the meiotic resumption after 22 h of culture, giving a higher percentage of GV-I stage oocytes and a lower percentage of GV-III stage oocytes in the ROS group than in A or B groups (Table 1). Just before culture and after treatment with roscovitine, oocytes were similar for both nuclear stages. The percentage of oocytes reaching the Met-I stage was higher in the B group than in the remaining three groups.
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, intracellular GSH content increased after oocytes were cultured. When oocytes were kept for 22 h in culture, GSH content increased from 2.24 to 10.74 pmol/oocyte, and still higher when roscovitine was present during culture (15.23 pmol/oocyte). After IVM, no differences were observed independently of a 22 h pre-maturation period with (ROS-IVM) or without (IVM) roscovitine, being 7.02 and 7.48 pmol/oocyte respectively. However, both groups showed a lower amount of GSH than the ROS group.
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After embryo transfer, both ROS-IVM and IVM sows from the second replicate became pregnant and delivered 12 (four male/eight female) and ten (five male/five female) healthy piglets respectively (Table 3). The weight of animals (means±S.E.M.), assessed 42 days after birth, was 8.3±0.5 and 11.5±0.6 kg respectively for ROS-IVM and IVM groups. Sows from replicates 1, 3 and 4 showed heat estrus 21–24 days after embryo transfer.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The effect of roscovitine on the resumption of meiosis has been shown previously in pigs. Ju et al.(2003) found that levels of 80–120 µmol/l roscovitine (Sigma, R-7772) were necessary to inhibit GVBD in 83–91% of oocytes. However, they assessed the nuclear stage of oocytes at 44 h from the beginning of culture in roscovitine, when the activity of the inhibitor could have been decreased in the medium (Meijer & Raymond 2003). In our study, doses of 50 µmol/l roscovitine were enough to block the GVBD in >90% of oocytes after 22 h of culture with similarities between control and ROS groups being evident for the GV-I and GV-III stages. In contrast, when the IVM medium without roscovitine was employed, oocytes started to resume meiosis spontaneously at this time. Moreover, when the NCSU-37 medium without dibutyryl cAMP was used, the progression to stages beyond GV-4 was faster, since the inhibitory effect of dibutyryl cAMP (Funahashi et al. 1997) was absent. These data first demonstrate that progression of porcine oocytes to the GV-III stage in the presence of 50 µmol/l roscovitine is inhibited in a different manner than in the presence of dibutyryl cAMP or in the absence of inhibitors, since they remain at similar nuclear stages to those in the follicles (as assessed just after recovery).
The reversibility of the roscovitine effect on the nuclear maturation has been corroborated in this study by the high proportions of Met II stage oocytes reached after 44 h of culture in the conventional IVM system following the 22 h of inhibition. However, it is well documented that nuclear and cytoplasmic maturation are not always correlated (Coy et al. 1999). The resumption of meiosis in oocytes after removal from roscovitine, presumably re-starting the activation of MPF, is not a prerequisite to ensure the cytoplasmic maturation of such oocytes. On the contrary, intracellular GSH content and the ability of oocytes to form a male pronucleus are commonly accepted parameters for assessing cytoplasmic maturation of porcine oocytes (Funahashi et al. 1995, Sawai et al. 1997, Coy et al. 1999). GSH is produced via the 
-glutamyl cycle and is dependent on the amount of cysteine available to the cell (Meister & Tate 1976). Cysteine is commonly added to pig oocyte maturation medium as a substrate for GSH synthesis to promote male pronuclear formation (Yoshida et al. 1993) at a 0.57 mmol/l concentration, and this was the case for NCSU-37. In this study, the GSH content of immature oocytes just after removal from follicles was lower than that found after 22 or 44 h of culture in IVM medium, the latter value being at the same baseline level as reported by Brad et al.(2003). The IVM system employed was presumably effective for oocyte GSH synthesis.
The GSH content was higher at 22 h of culture than at 44 h, in contrast to the findings of Yoshida et al.(1993) who have reported a continuous increase in GSH content during IVM. On the one hand, these authors employed TCM-199 for IVM, which is a rich culture medium with readily available GSH precursors (cysteine, cystine, glutamic acid, glutamine and glycine) and even glutathione. However, in our study NCSU-37 was used and was supplemented only with cysteine and glutamine (glutamine is the first cystine pre-cursor auto-oxidized, within 1 h, in the maturation medium (de Matos & Furnus 2000) that cannot be incorporated so easily as cysteine (Yoshida & Takahashi 1998)). On the other hand, it has been reported that GSH synthesis during initial and mid phases of porcine oocyte maturation is related to the acquisition of sperm nuclear decondensing ability (Yoshida 1993) and GSH synthesis during the initial stages of hamster oocyte maturation is responsible for the higher GSH levels in mature oocytes (Perreault et al. 1988). These observations suggest a strong GSH synthesis during the first half of oocyte maturation and would be in agreement with our results showing a GSH peak after 22 h of culture.
The present study also shows that oocytes pre-cultured in roscovitine for 22 h had a higher level of GSH than those cultured with dibutyryl cAMP for the same time. Cystine uptake activity is abolished by the mechanical disruption of gap junctional communication (Yoshida & Takahashi 1998), and GSH synthesis may be impaired due to the uncoupling of cumulus cells (de Matos et al. 1997). Since roscovitine-treated oocytes do not show cumulus expansion as shown by dibutyryl cAMP-treated oocytes (Marchal et al. 2001, Coy et al. 2004, and personal observations in the present experiments), the closest cooperation between cumulus cells and oocyte would be maintained for a longer time, explaining in part the higher GSH content observed in roscovitine-treated oocytes. Also, the inhibitory effect of roscovitine must be considered not only on the cdk but also on RNA synthesis (Ljungman & Paulsen 2001). Because GSH is involved in multiple functions, including DNA and protein synthesis (Lafleur et al. 1994), it could be expected that oocytes treated with roscovitine showed a higher GSH content than their counterparts, as was observed in our case since this functional activity of GSH was probably decreased.
Regarding male pronuclear formation, our data first show that roscovitine does not affect this parameter. This result was expected due to the similar levels of GSH found for in vitro-matured oocytes pre-cultured or not with roscovitine, since it has been reported that male pro-nuclear formation is related to intracellular GSH content (Yoshida et al. 1992, Funahashi et al. 1995). Moreover, no differences were found for any of the fertilization parameters assessed at 18 hpi, in agreement with most previous studies in bovine (Mermillod et al. 2000, Ponderato et al. 2001, Coy et al. 2005).
Finally, the key result in our study was the pregnancy of one recipient sow and the delivery of 12 healthy piglets when roscovitine-pre-cultured embryos were transferred. It is well documented that the in vitro production of pig embryos with the ability to develop to term is difficult due to polyspermy and deficient embryo culture media (Coy & Romar 2002, Funahashi 2003). Only 20–30% of transferred embryos survive despite considerable improvement in the techniques for in vitro production of porcine embryos (Abeydeera 2002), and approximately six piglets per sow after surgical embryo transfer are obtained (Abeydeera et al. 1998, Kikuchi et al. 1999). With these premises, and taking into account that about 37 and 41% of the transferred zygotes in IVM and ROS-IVM could be expected to develop to term, a high number of putative embryos was transferred at only 22–24 h of insemination and in vitro culture, in order to ensure a sufficient number of viable embryos in the oviduct of the synchronized sow. Thus, a final success of 25% for both IVM and ROS-IVM groups could be considered satisfactory and is among the normal rates reported in the literature.
In conclusion, this study demonstrates that pig oocytes cultured for 22 h with 50 µmol/l roscovitine remained at similar nuclear stages to oocytes just after recovery. Cytoplasmic maturation assessed as intracellular GSH content and male pronuclear formation were not affected detrimentally by culture with roscovitine. Moreover, embryos obtained from this two-step IVM system can be developed to term showing the final competence of porcine oocytes pre-cultured with roscovitine, thus making its introduction desirable in assisted reproductive technologies programs.
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Acknowledgements |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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