In vitro maturation of pig oocytes with different media, hormone and meiosis inhibitors

Animal Reproduction Science 97 (2007) 375–381

In vitro maturation of pig oocytes with different media, hormone and meiosis inhibitors Mariana Groke Marques ∗ , Alessandra Corallo Nicacio, Viviane Purri de Oliveira, Anibal Ballarotti Nascimento, Heloisa Vasconcelos Amaral Caetano, Camilla Mota Mendes, Marco Roberto Bourg Mello, Marcella Pecora Milazzotto, Mayra Elena Ortiz D‘Avila Assumpc¸a˜ o, Jos´e Antˆonio Visintin Department of Animal Reproduction, Faculty of Veterinary Medicine of the University of S˜ao Paulo, Brazil Received 24 October 2005; received in revised form 15 February 2006; accepted 27 February 2006 Available online 4 April 2006

Abstract This study evaluated in vitro maturation (IVM) of pig oocytes in two maturation media (TCM199 and NCSU23) supplemented with 10% porcine follicular fluid (pFF) or 0.1% polyvinyl alcohol (PVA) and four hormonal treatments. The best media was then used to evaluate the effect of reversible meiosis inhibitors cycloheximide (CHX) (5 ␮M) and butyrolactone I (12.5 ␮M) on the maturation of pig oocytes was evaluated. After maturation for 44 h, the oocytes were fixed, stained and examined under epifluorescence microscopy. The comparison of the proportion of oocytes in metaphase II (MII) revealed that hormonal Treatment 2 (incubation for 22 h–10 ng EGF/ml, 10 IU hCG/ml and 10 IU eCG/ml, followed by incubation for 22 h–10 ng EGF/ml) presented higher repeatability percentages: TCM + PVA (54.5%, 61/112); TCM + pFF (65.0%, 63/97); NCSU23 + PVA (54.6%, 65/119) and NCSU23 + pFF (58.1%, 61/105). The comparison of maturation media showed that TCM199 presented more constant results than NCSU23. Regarding supplementation with pFF or PVA, TCM199 with pFF presented better results. The comparison between butyrolactone I and cycloheximide demonstrated that both drugs effectively inhibited meiosis; however, only cycloheximide presented metaphase II percentages similar to the control (70.29% and 75.49%, respectively). In conclusion, it is recommended the use of TCM199 medium supplemented with pFF and hormonal treatment with 10 ng EGF/ml, 10 UI hCG/ml and 10 UI eCG/ml during the first 22 h and more 22 h with 10 ng EGF/ml for the pig

∗

Corresponding author. Tel.: +55 11 30917916; fax: +55 11 30917412. E-mail addresses: [email protected] (M.G. Marques), fulo [email protected] (A.C. Nicacio), [email protected] (V.P.d. Oliveira), [email protected] (A.B. Nascimento), [email protected] (H.V.A. Caetano), [email protected] (C.M. Mendes), [email protected] (M.R.B. Mello), [email protected] (M.P. Milazzotto), [email protected] (M.E.O.D. Assumpc¸a˜ o), [email protected] (J.A. Visintin). 0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.02.013

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oocytes maturation. Butyrolactone I and cycloheximide effectively arrested/resumpted maturation; however, the oocytes percentages in metaphase II was the same for both cycloheximide and the control groups. © 2006 Elsevier B.V. All rights reserved. Keywords: Oocyte; Oocyte maturation; Pig; Butyrolactone I; Cycloheximide

1. Introduction Oocyte maturation is one of the most important stages for in vitro production of embryos. In addition to undergoing nuclear modifications (nuclear maturation) to develop fertilization capacity, oocytes store substances and undergo morphological alterations (cytoplasmic maturation), which promote and are essential for the early development of the embryo. Since in vitro maturation (IVM) is not as efficient as in vivo maturation, new studies are needed to clarify optimal culture media combinations needed to maximise maturations rates. Several media support in vitro nuclear maturation of pig oocytes but not cytoplasmic maturation. Problems in cytoplasmic maturation interfere with the formation of the pronuclei after penetration of the sperm, despite normal germinal vesicle (GV) breakdown and extrusion of the first polar body (Abeydeera, 2002). Based on observations of maturation media supplemented with pFF, Yoshiba et al. (1992) concluded that porcine follicular fluid (pFF) contains substances that improve the expansion of the cells of the cumulus oophorus, nuclear maturation and normal fertilization. Because pFF contains high concentrations of superoxide dismutase, it also plays an important role in the protection of oocytes against oxidative stress (Tatemoto et al., 2004). Superoxide dismutase increases cytoplasmic maturation and improves development competence after fertilization. Nevertheless, pFF introduces a series of unknown factors to the medium, which cause difficulties in technical standardization and in the exact identification of the substances that are essential to the regulation of maturation (Yoshiba et al., 1992). As an alternative, researchers have been developing new maturation media supplemented with polyvinyl alcohol (PVA), as a substitute for pFF. However, in order to be effective, media using PVA should be supplemented with growth factors such as epidermal growth factor (EGF) (Abeydeera et al., 2000; Kishida et al., 2004) and/or essential and nonessential amino acids (Hong et al., 2004). The main beneficial effect of EGF is to stimulate the synthesis of intracellular glutathione (Abeydeera et al., 2000). In addition to the beneficial effect on embryo development, glutathione protects the DNA, acts on the protein synthesis and amino acid transport, and promotes male pronuclear formation (Whitaker and Knight, 2004). In the ovary, mammalian oocytes are arrested in the initial stage of meiotic division (germinal vesicle) until the release of the pre-ovulatory gonadotrophin that stimulates the resumption of meiosis (Richard and Sirard, 1996). During the final stage of maturation, immature oocytes develop from germinal vesicle (prophase I) to metaphase II (MII). At this stage, meiosis is again arrested, and this second arrest is normally interrupted by fertilization (Lonergan et al., 2000). The use of a two-step culture system (arrest/resumption), by means of the reversible inhibition of GV breakdown in bovine oocytes, results in a longer maturation period, allowing oocytes to develop complete meiotic competence (maturation of the nucleus and cytoplasm) (Kubelka et al., 2000). GV breakdown inhibition is possible through the inhibition of metaphase promoting factor (MPF). This is a protein complex composed of two main subunits, one a cyclin dependent kinase (p34cdc2 ) and the other a cyclin (Alberts et al., 1994).

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Experiments have shown that butyrolactone I is a selective inhibitor of cyclin dependent kinases arresting the cellular cycle in G1/S and G2/M. In mammalian oocytes (rats, pigs and cattle) butyrolactone I inhibits the germinative vesicle breakdown (Motlik et al., 1998) and is a direct inhibitor of the MPF, because it competes with ATP for the binding site p34cdc2 and inhibits the dephosphorilation that is essential to activate MPF (Wu et al., 2002). In their studies with bovines, Pavlok et al. (2000) demonstrated that when oocytes in culture medium are exposed to butyrolactone, RNA synthesis continues and is only reduced with the compactation of the nucleus. Cycloheximide (CHX) is a protein synthesis inhibitor in eukaryotic cells and could be used to block the GV breakdown (Beux et al., 2003). Lonergan et al. (1998) demonstrated that when the maturation of bovine oocytes is arrested with CHX, oocytes presented competent development after resumption and were subjected to maturation, fertilization and in vitro culture. Based on these data, this study evaluated 10% pFF or 0.1% PVA supplementation on TCM199 and NCSU23 maturation media with four different hormonal treatments. In order to determine better conditions for in vitro maturation of pig oocytes, this study also evaluated the arrest/resumption of oocyte maturation with butyrolactone I and cycloheximide. 2. Material and methods 2.1. Collection of ovaries, follicle aspiration and selection of oocytes Abattoir-derived porcine ovaries were transported to the laboratory between 25 and 28 ◦ C. Follicles measuring from 2 to 5 mm were aspirated using an 18 gauge needle attached to a 10 ml syringe, and follicular fluid was transferred into 50 ml conic tubes for sedimentation. Oocytes presenting a thick and intact cumulus oophurus layer, and cytoplasm with homogeneous granulations were selected for IVM. 2.2. Experiment 1: maturation in different media and hormonal treatments Cumulus-oocyte complexes (COCs) were divided into 16 groups (2 media group × 2 supplementation × 4 hormonal treatments). The two maturation media were: TCM199 media (supplemented with 3.05 mM glucose, 0.91 mM sodium pyruvates, 50 IU/ml gentamycine and 0.57 mM cysteine) and NCSU23 media (supplemented with 50 IU/ml gentamycine and 0.57 mM cysteine); both supplemented with 10% pFF or 0.1% PVA, and four hormonal treatments. Hormonal treatments were: Treatment 1 (22 h in maturation medium with 10 UI eCG/ml and 10 UI hCG/ml and 22 h without hormone); Treatment 2 (22 h in maturation medium supplemented with 10 ng EGF/ml, 10 UI hCG/ml and 10 UI eCG/ml and 22 h supplemented with 10 ng EGF/ml); Treatment 3 (22 h in maturation medium supplemented with 10 UI eCG/ml and 22 h supplemented with 10 UI hCG/ml); Treatment 4 (22 h in maturation medium supplemented with 10 ng EGF/ml and 10 UI eCG/ml and 22 h supplemented with 10 ng EGF/ml and 10 UI hCG/ml). In vitro maturation was performed in an incubator at 39 ◦ C, in an atmosphere of 5% CO2 in air and high humidity. 2.3. Experiment 2: arrest and resumption of nuclear maturation In this study, oocytes were maintained for 10 h on the best maturation media chosen from Experiment 1 (TCM199 + 10% pFF + 10 ng EGF/m + 10 UI hCG/ml + 10 UI eCG/ml) added with butyrolactone I (12.5 ␮M) or cycloheximide (5 ␮M). After this period, oocytes underwent maturation (22 h in maturation medium supplemented with 10 ng EGF/ml, 10 UI hCG/ml and 10 UI

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eCG/ml and 22 h supplemented with 10 ng EGF/ml) proposed on Experiment 1. Control group was submitted to the maturation protocol for 44 h without meiosis inhibitions. Oocytes were assessed to determine the percentage of arrest (0 h control, 10 h control, 10 h butyrolactone I and 10 h cycloheximide) and maturation rate after resumption (44 h control, 44 h butyrolactone I and 44 h cycloheximide). Oocyte incubation was performed in an incubator at 39 ◦ C, in an atmosphere of 5% CO2 in air and high humidity. 2.4. Examination of metaphase II percentage After maturation (44 h), oocytes from both Experiments 1 and 2 were denuded of cumulus oophorus cells by exposure to hyaluronidase (5 mg/ml in PBS without calcium and magnesium) and by mechanical process. Oocytes were fixed using Triton 100-X in paraformaldehyde 3.7% for one hour and incubated in PBS with 0.3% BSA and Triton 100-X for another hour. After fixation, the oocytes were mounted on glass slides and coverslips using one drop of 10 (g Hoechst 33,342 ml−1 of glycerol and examined under microscopy (Zeiss) with epifluorescence (filter of maximum excitation of 365 nm and maximum emission of 480 nm) to evaluate nuclear maturation rates. 2.5. Statistical analysis Assessment of results (percentages) was performed using the Equality of two ratios Test (Daniel, 1995) with a 5% significance level. 3. Results 3.1. Experiment 1: maturation in different media and hormonal treatments Table 1 illustrates the distribution of metaphase II oocytes in the different maturation media and hormonal treatments. Considering of maturation media showed that TCM199 presented more constant results than NCSU23. Comparison of pFF and PVA supplementation showed that TCM199 supplemented with pFF presented better results. Comparison of hormonal treatments showed that Treatment 2 was the only treatment with no variation, regardless of medium or supplementation. Therefore, TCM199 with pFF supplemented with hormonal Treatment 2 was the Table 1 Oocytes percentage in metaphase II in the different maturation media and hormonal treatments Hormonal treatments

Maturation medium TCM + PVA % (n) (56/113)Aa

1

49.6

2

54.5 (61/112)Aa

3 4

47.7 (53/111)Aa 43.8 (53/121)Aac

TCM + pFF % (n) 69.0

(89/129)Ab

66.4 (85/128)Ab 75.2 (82/109)Ab

NCSU23 + PVA % (n) 53.4

(62/116)Aa

NCSU23 + pFF % (n) 66.2 (90/136)Ab

54.6 (65/119)Aa

58.1 (61/105)ABa

63.8 (67/105)Ab 39.3 (46/117)Ba

65.1 (71/109)ABb 52.9 (73/138)Bc

Different superscript letters (A and B) in each maturation media (column) present statistical significant differences (P < 0.05); different superscript letters (a–c) in each hormonal treatment (line) present significant difference (P < 0.05).

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Table 2 Oocyte percentages in different development stages: GV, GVBD and MI in groups control 0 h (0 h), control 10 h (10 h of maturation), butyrolactone I 10 h (10 h of arrest) and cycloheximide 10 h (10 h of arrest) Maturation stages

Control 0 h % (n/total)

Control 10 h % (n/total)

Butyrolactone 10 h % (n/total)

Cyclohexemide 10 h % (n/total)

GV GVBD MI

54.3 (19/35)Aa 42.8 (15/35)Aa 2.9 (1/35)Ba

20.0 (4/20)Ab 80.0 (16/20)Bb 0.0 (0/20)Ca

45.7 (16/35)Aab 51.4 (18/35)Aa 2.9 (1/35)Ba

45.5 (15/33)Aab 54.5 (18/33)Aab 0.0 (0/33)Ba

Different superscript letters (A–C) in each maturation period (column) represent significant difference (P < 0.05); different superscript letters (a and b) in each maturation stage (line) represent significant difference (P < 0.05). GV, germinative vesicle; GVBD, germinative vesicle breakdown; MI, metaphase I. Table 3 Oocyte percentages in each stage of the cellular cycle in groups under maturation arrest and control group Maturation Stages

Butyrolactone I % (n/total) (1/191)Aa

GV VGBD MI ANA TEL

0.52 0 (0/191)Aa 36.13 (69/191)Ba 0 (0/191)Aa 0 (0/191)Aa

MII

63.35 (121/191)Ca

Cycloheximide % (n/total) (2/175)Aa

1.14 0 (0/175)Aa 28.57 (50/175)Bab 0 (0/175)Aa 0 (0/175)Aa

Control % (n/total) 0.98 (1/102)Aa 1.96 (2/102)Aa 18.64 (19/102)Bb 0.98 (1/102)Aa 1.96 (2/102)Aa 75.49 (77/102)Cb

Different superscript letters (A–C) in each maturation group (column) present significant difference (P < 0.05); different superscript letters in (a and b) each maturation stage (line) present significant difference (P < 0.05). GV, germinative vesicle; V, germinative vesicle breakdown; MI, metaphase I; ANA, anaphase; TEL, telophase; MII, metaphase II.

most suitable maturation media for Experiment 1, and therefore, chosen as the main media for Experiment 2. 3.2. Experiment 2: arrest and resumption of nuclear maturation The assessment of maturation arrest (Table 2) showed that butyrolactone I and cycloheximide were effective in arresting oocytes, because both had the same germinative vesicle (GV) percentages: control 0 h (54.3%), butyrolactone I 10 h (45.7%) and cycloheximide 10 h (45.5%). However, butyrolactone I and cycloheximide did not arrest all oocytes, since the results are similar to the results obtained for the 10 h control group. Examination of the number of oocytes in metaphase II in the control, butyrolactone and cycloheximide groups after 44 h of maturation (Table 3) showed that both blockage agents were efficient as regards the production of mature oocytes. Maturation after arrest for the cycloheximide group did not differ from the control and butyrolactone I groups (70.29%, 75.49% and 63.35%, respectively). Nevertheless, a significant difference was observed between butyrolactone I and control groups. 4. Discussion Analysis of the results presented in Table 1, showed that maturation media, pFF and PVA supplementations, and hormonal treatments proved efficient in the maturation of pig

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oocytes. As expected, supplementation with pFF was more efficient than supplementation with PVA. In this study, MII percentage did not improve with EGF, which is in agreement with Abeydeera et al. (2000) who describe that, although mature oocytes in EGF medium do not present higher MII percentage, they present higher blastocyst rates, because EGF promotes cytoplasmic maturation, consequently improving embryo development. Hormonal Treatment 2 was chosen because when added to TCM199 and NCSU23 media with pFF or PVA supplementation, it presented more constant results, and therefore it was used as the standard hormone treatment in subsequent experiments. In this experiment, NCSU23 medium presented variation between the hormonal treatments. Since this variation was not observed with TCM199, it was chosen as the standard media for subsequent experiments. From the results of her studies, Lima (unpublished results) concluded that hormonal supplementation with eCG and hCG for 48 h of maturation reduced the nuclear maturation rates in comparison to hormonal supplementation with eCG and hCG for 24 h and no hormones for another 24 h (TCM199 = 65.41% and 46.09% and NCSU23 = 61.31% and 46.28%, respectively). In this study, it was noticed no difference between hormonal supplementation for 44 or 24 h. However, it was used hormones individually (first 22 h with eCG followed by 22 h with hCG). This suggests that eCG might be harmful after the first 22 h of maturation. Nevertheless, hCG could be used during the 44 h of oocyte maturation. It was evaluated nuclear maturation only. Funahashi and Day (1993) demonstrated that 20 h of maturation with no hormonal supplementation would be necessary to improve cytoplasmic maturation. Therefore, more studies are needed to determine the role of these hormones in nuclear and cytoplasmic in vitro maturation of pig oocytes. Our study suggests that environment conditions might be affecting the premature breakdown of the germinative vesicle. At 0 h of maturation (Table 2) was obtained 54.3% GV, a result different from that described by Ye et al. (2002) who identified 95% of oocytes in GV in the control group. In this study, both inhibitors were effective in the production of MII oocytes (63.35% and 70.29%; butyrolactone I and cycloheximide, respectively). In their studies, Ye et al. (2002) demonstrated that 80% of oocytes with maturation arrested by cycloheximide reached MII after resumption. According to Wu et al. (2002), 83.4% of oocytes submitted to arrest by butyrolactone I reach MII after resumption without the inhibitor. The reduced maturation rates obtained in this study after maturation might be explained by the rate of oocytes in GV at 0 h. After GVBD, maturation arrest loses its beneficial effect in increasing RNA synthesis. This factor might explain the reason that MII percentages after resumption were lower than those described in literature. In conclusion, it is recommended the use of TCM199 medium supplemented with pFF and hormonal treatment with 10 ng EGF/ml, 10 UI hCG/ml and 10 UI eCG/ml during the first 22 and more 22 h with 10 ng EGF/ml for pig oocytes maturation. Butyrolactone I and cycloheximide effectively arrested/resumpted maturation; however, the oocytes percentages in metaphase II was the same for both cycloheximide and the control groups. More studies are needed to verify if reversible arrest of meiosis with butyrolactone I and cycloheximide improve cytoplasmic maturation, increasing the storage of substances needed for the initial development of the embryo, and consequently improving the rate of in vitro fertilization and blastocyst yield. Acknowledgements The authors thank Melanie Klemm for the translation and revision of the text. Support was also provided by FAPESP.

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