Reprod Dom Anim doi: 10.1111/rda.12313 ISSN 0936–6768
Mitochondrial Patterns in Bovine Oocytes with Different Meiotic Competence Related to Their in vitro Maturation M Jeseta1, D Ctvrtlikova Knitlova1, K Hanzalova1, P Hulinska1, S Hanulakova1, I Milakovic1, L Nemcova2, J Kanka2 and M Machatkova1 1 Veterinary Research Institute, Brno, Czech Republic; 2Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Libechov, Czech Republic
Contents This study was designed to specify chromatin and mitochondrial patterns in bovine oocytes with different meiotic competence in relation to maturation progress, resumption of meiosis, MII onset and completion of maturation. Oocytes with greater or lesser meiotic competence, recovered separately from medium (MF) and small follicles (SF), were categorized according to morphology. Four oocyte categories, healthy and light-atretic MF and healthy and light-atretic SF oocytes were matured and collected at 0, 3, 7, 16 and 24 h of maturation. Specific differences in terms of chromatin and mitochondrial patterns were found among the maturing oocyte categories. Resumption of meiosis was accelerated in light-atretic oocytes, as compared with healthy oocytes, regardless of their meiotic competence. More competent oocytes activated mitochondria twice during maturation, before resumption of meiosis and before completion of maturation, while less competent oocytes did it only once, before completion of maturation. Changes in mitochondrial activity differed in light-atretic compared with healthy in both more and less competent oocytes. Healthy meiotically more competent oocytes formed clusters and produced ATP for the whole time of maturation until its completion, while light-atretic more competent oocytes and healthy less competent oocytes reduced these activities earlier, at MII onset. Contrary to these oocyte categories, light-atretic less competent oocytes increased cluster formation significantly before resumption of meiosis. It can be concluded that bovine oocytes with different meiotic competence and health differed in the kinetics of mitochondrial patterns during maturation.
Introduction At the present time, the developmental competence of bovine oocytes is a limiting factor for wider application of assisted reproductive technologies in cattle breeding. Even though maturation is close to optimum and fertilization is effective, development of fertilized oocytes to transferable embryos is not adequate. To improve embryo development, enough knowledge of the intrinsic quality of both immature and mature oocytes is required. It is generally known that the developmental ability of an embryo is determined by an initial potential of the oocyte, which reflects follicular conditions at the time of its recovery. In this context, the meiotic and developmental competence of the oocyte can be expressed by both size and health of the follicle, and its status can be assessed on the basis of cumulus– oocyte morphology (Sirard and Blondin 1996). Oocytes with the first signs of atresia and a less compact cumulus originating from larger or early-atretic follicles are more developmentally competent than those from smaller or © 2014 Blackwell Verlag GmbH
late-atretic follicles (Blondin and Sirard 1995; Sirard and Blondin 1996; Feng et al. 2007). Oocyte maturation is accompanied by major changes in the ooplasm, which lead to the completion of nuclear and cytoplasmic maturation (Brevini et al. 2007). In both processes, as well as in fertilization and early embryo development, mitochondria play an important role in matching adenosine triphosphate (ATP) supply to demand (Bavister and Squirrell 2000; Sathananthan and Trounson 2000; Brevini et al. 2005; Tarazona et al. 2006; Van Blerkom and Davis 2007; Van Blerkom 2008). Mitochondria in oocytes and embryos are important not only for cellular ATP production but also other functions, such as participation in calcium and redox signalling (Van Blerkom 2011). They regulate cellular calcium homeostasis and ROS defence which are critical to cell survival and embryonic development (Dumollard et al. 2006; Cao and Chen 2009). Mitochondrial dysfunction with low ATP production is one of the major factors compromising oocyte quality (Dumollard et al. 2006; Yu et al. 2010). Mitochondria are also known to be involved in oocyte atresia (Torner et al. 2004; Wang et al. 2009). The leakage of cytochrome C from dying mitochondria is a key event in the cascade leading to apoptosis (Shimizu et al. 1999). For these reasons, mitochondrial patterns can be taken as markers of the quality and developmental capacity of mammalian oocytes and embryos (Nagai et al. 2006; Egerszegi et al. 2010). However, two aspects are important for assessment of mitochondria: their quality and quantity (Cummins 2004). The morphology of mitochondria (Van Blerkom 2004), their aggregation (Torner et al. 2004) and gene expression (Kanka et al. 2012) have been described in relation to the developmental potential of oocytes. Mitochondrial aggregates with the smooth endoplasmic reticulum (SER), which are probably involved in Ca2+ release from SER and subsequent energy production, have been found in human oocytes matured in vivo (Van Blerkom 2011). Mitochondrial clusters are usually used as a criterion of cytoplasmic quality in oocytes matured in vitro (KatskaKsiazkiewicz et al. 2011). Although the importance of mitochondria for oocyte development is known and is likely to increase in the future (Wang et al. 2009), there is still a lack of information about their functional changes during oocyte maturation. In our previous study, we concluded that, before maturation, meiotically more competent oocytes had
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M Jeseta, D Ctvrtlikova Knitlova, K Hanzalova, P Hulinska, S Hanulakova, I Milakovic, L Nemcova, J Kanka and M Machatkova
more active and reorganized mitochondria than meiotically less competent oocytes. Although the less competent oocytes showed an increase in mitochondrial activity after maturation, they remained inferior to more competent oocytes in both cluster formation and ATP production (Machatkova et al. 2012). This study was designed to characterize mitochondrial patterns in bovine oocytes with different meiotic competence in the course of maturation, related to the resumption of meiosis, MII onset and completion of maturation.
Materials and Methods All chemicals used in this study were purchased from Sigma-Aldrich Chemical Co. (Prague, Czech Republic) unless otherwise stated. Oocyte collection and categorization Slaughtered Holstein dairy cows (total no = 131), aged 4–6 years, with a checked ovarian cycle stage, were used in the experiments. Ovaries in growth/stagnation and regression phases of folliculogenesis, assessed by follicular and corpus luteum (CL) morphology, were used for oocyte collection. The oocytes were recovered separately from medium (MF; 6–10 mm) and small (SF; 2–5 mm) follicles by aspiration and slicing, respectively. The cumulus–oocyte complexes (COC) suitable for IVM and IVF were selected and categorized as healthy (homogenous cytoplasm without dark spots and compact homogenous cumuli) or light-atretic (slightly granulated cytoplasm with ≤ two dark spots and expanded outer layers of cumuli) according to the Blondin and Sirard classification (Blondin and Sirard 1995). Four oocyte categories, healthy MF, light-atretic MF, healthy SF and light-atretic SF, were used in the experiments. Oocyte maturation The four oocyte categories were matured separately in TCM-199 medium (Earle0 s salts), supplemented with 20 mM sodium pyruvate, 50 IU/ml penicillin, 50 lg/ml streptomycin, 5% oestrus cow serum (ECS; Sevapharma, Prague, Czech Republic) and gonadotropins (P.G. 600 15 IU/ml; Intervet, Boxmeer, Holland) in four-well (a)
plates (Nunclon Intermed, Roskilde, Denmark) at 38.8°C under 5% CO2 in air. An adequate part of oocytes of each category was recovered at 0, 3, 7, 16 and 24 h of maturation (hm), denuded of cumulus cells and examined. Oocyte staining Mitochondria were stained in PBS supplemented with 0.4% BSA and 200 nM MitoTracker Orange CMTM Ros dye (Molecular Probes, Eugene, OR, USA) for 30 min at 38.8°C. Only respiring mitochondria were stained with this cell-permeant mitochondrial-specific dye. After washing, the oocytes were fixed in 3.7% paraformaldehyde for 60 min at room temperature. They were washed in PBS and mounted on glass slides, avoiding oocyte compression, using Vectashield medium (Vector Lab, Burlingame, CA, USA) containing 1 lM of DNA dye (SYTOX Green; Invitrogen; Carlsbad, CA, USA) specific for dyeing of chromatin. The slides were stored below 0°C until examined. Oocyte examination The oocytes were examined with the use of a laser scanning confocal microscope (Leica TCS SP2 AOBS; Leica, Heidelberg, Germany) equipped with Ar and DPSS lasers. The 488-nm excitation band and 490- to 515-nm detector and the 561-nm excitation band and 565- to 600-nm detector were used for chromatin and mitochondria visualization, respectively. The 40 9 Leica HCX PL APO CS objective, pinhole, offsets, gain and AOBS were adjusted. These parameters were kept throughout the whole experiment. The oocytes were scanned in equatorial optical sections, and microphotographs were saved and processed using the NIS-ELEMENTS AR 3.0 software (Laboratory Imaging, Prague, Czech Republic). Chromatin and mitochondrial status assessment A total of 1180 oocytes were evaluated for nuclear maturation and mitochondrial status. The GV, GVBD and MII rates, mitochondrial activity, expressed as a mean fluorescence per oocyte, and a frequency of (b)
Fig. 1. Representative images of oocytes without (a) or with (b) mitochondrial clusters demonstrated by confocal microscopy. Oocyte was stained by MitoTracker Orange CMTM Ros. Scale bar represents 20 lm
© 2014 Blackwell Verlag GmbH
Kinetics of the Mitochondrial Pattern in Bovine Oocytes
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Tukey’s post hoc test using ANOVA SPSS version 11.5 for Windows (SPSS, Inc. Chicago, IL, USA).
Table 1. Nuclear maturation in the oocyte categories at different maturation intervals Hours of maturation (hm) 0
Oocytes MF Healthy Light-atretic SF Healthy Light-atretic
3
7
16
Chromatin configuration rate (%) GV GVBD MII
n
GV
194 160
100.0a 100.0a
97.6a 100.0a
26.8a 51.4b,c
13.3a 35.0b
412 414
100.0a 98.3a
100.0a 100.0a
46.4b 66.1c
19.4a,b 24.0b
Results
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Comparison of the oocyte categories The four oocyte categories were compared in terms of nuclear maturation, mitochondrial activity, mitochondrial cluster formation and ATP production in the selected maturation intervals (Tables 1–4). A comparison of chromatin configuration showed that, in MF oocytes, the GVBD rates at 7 hm and MII rates at 16 hm were significantly higher (p < 0.05) in light-atretic than in healthy oocytes. At 24 hm, however, the maximum MII rate was achieved by both healthy and light-atretic MF oocytes. In SF oocytes, a significantly higher GVBD rate at 7 hm and a higher MII rate at 16 hm were found in light-atretic oocytes, as compared with healthy oocytes. At 24 hm, the MII rate of light-atretic oocytes had the lowest value of all oocyte categories (Table 1). Mitochondrial activity was significantly higher in healthy and light-atretic MF and healthy SF oocytes up to 7 hm, as compared with light-atretic SF oocytes (p < 0.05). From this time on, there were no differences in mitochondrial activity among the four oocyte categories (Table 2). An increased frequency of oocytes with clusters was observed in light-atretic SF oocytes at 7 hm. At 24 hm,
MII
100.0a 100.0a 93.5a,b 84.6b
Values with different superscripts within the same column are significantly different (p < 0.05).
oocytes with mitochondrial clusters were evaluated in the four oocyte categories. The morphology of oocytes with the presence or absence of clusters is shown in Fig. 1a,b, respectively. ATP content assessment A total of 210 oocytes were examined for ATP content using a bioluminescent somatic cell assay kit (FLASC). Briefly, the oocytes were rinsed three times in Ca2+- and Mg2+-free PBS and transferred individually, in 50 ll of solution, into plastic tubes. After adding 100 ll ice-cold somatic cell reagent (FLSAR) to each tube, the oocytes were incubated on ice for 5 min. Then, 100 ll ice-cold assay mix (FLAAM assay mix and FLAAB dilution buffer; 1:25) was added to each tube. The tubes were kept for an additional 5 min at room temperature in darkness. The intensity of luminescence (luciferin–luciferase reaction) was measured using Luminoskan (type 391A; Labsystems, Helsinki, Finland). A nine-point standard curve was determined, and a negative control was run for every 20 oocytes. The ATP content per oocyte was calculated by the formula derived from a linear regression of the standard curve in the four oocyte categories.
Table 3. Frequency of oocytes with mitochondrial clusters in the oocyte categories at different maturation intervals Frequency of oocytes with clusters (%) Hours of maturation (hm) Oocytes MF Healthy Light-atretic SF Healthy Light-atretic
Statistical analysis At least four replicates were carried out for each oocyte category and each time interval. The results were analysed with the chi-square test, Student’s t-test and
n
0
3
7
16
24
190 157
0.0 0.0
14.63a 13.63a
17.07a,b 14.29a
29.27a 26.32a
57.50a 35.48b
310 330
0.0 0.0
16.67a 12.96a
19.60a,b 25.93b
32.80a 29.94a
41.70b 43.08b
Values with different superscripts within the same column are significantly different (p < 0.05).
Table 2. Mitochondrial activity in the oocyte categories at different maturation intervals Mitochondrial activity per oocyte (mean SD) Hours of maturation (hm) Oocytes MF Healthy Light-atretic SF Healthy Light-atretic
n
0
3
7
16
24
166 148
30.1 11.2a 25.8 13.1a
22.3 13.5a,b 21.2 18.0a,b
42.6 12.8a 32.3 15.0a,b
16.4 9.9a 27.7 14.7a
29.9 22.4a 32.2 15.0a
257 307
28.4 21.2a 8.5 3.4b
30.8 13.8a 8.3 5.8b
29.4 11.8a,b 15.0 9.1b
21.1 14.6a 21.2 17.0a
36.7 20.8a 36.8 15.1a
Values with different superscripts within the same column are significantly different (p < 0.05).
© 2014 Blackwell Verlag GmbH
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M Jeseta, D Ctvrtlikova Knitlova, K Hanzalova, P Hulinska, S Hanulakova, I Milakovic, L Nemcova, J Kanka and M Machatkova
Table 4. ATP content in the oocyte categories at different maturation intervals ATP content per oocyte in pmol (mean SD) Hours of maturation (hm) Oocytes MF Healthy Light-atretic SF Healthy Light-atretic
n
0
3
7
16
24
115 103
2.70 0.48a 2.52 0.59a
2.25 0.24a 1.64 0.46a
2.33 0.36a 2.22 0.33a
2.71 0.29a 2.61 0.28a
3.30 0.34a 2.79 0.35a
296 279
2.31 0.45a 2.38 0.53a
2.13 0.35a 1.97 0.39a
2.10 0.31a 2.22 0.35a
2.76 0.33a 2.67 0.48a
3.01 0.39a 2.84 0.43a
Values with the same superscripts within the same column are not significantly different.
a significantly higher frequency of oocytes with clusters (p < 0.05) was found in healthy MF oocytes than in the other three categories (Table 3). No significant differences in ATP content were observed among the oocyte categories in any maturation interval (Table 4). Course of changes in the oocyte categories To characterize mitochondrial pattern during maturation, the changes in mitochondrial activity, frequency of oocytes with clusters and ATP content were expressed as curves, and a comparison of values for maturation times in each oocyte category was made. The results are presented in Figs 2–4. Mitochondrial activity changes during maturation differed between MF and SF oocytes (Fig. 2a,b). In healthy MF oocytes, there was a significant increase (p < 0.05) from 3 to 7 hm and a significant decrease from 7 to 16 hm. In light-atretic MF oocytes, the curve was affected by atresia (Fig. 2a). In SF oocytes, a significant increase in mitochondrial activity in both healthy and light-atretic oocytes was found only from 16 to 24 hm (Fig. 2b). The frequency of oocytes with clusters increased in both healthy and light-atretic MF and SF oocytes (Fig. 3a,b). In healthy MF oocytes, a significant increase (p < 0.05) was observed from 7 to 16 hm and from 16 to 24 hm, while, in light-atretic MF oocytes, it was only between 7 and 16 hm (Fig. 3a). In healthy SF oocytes, a significant increase (p < 0.05) was observed from 7 to 16 hm. On the other hand, in light-atretic SF oocytes, the frequency of oocytes with clusters significantly increased earlier, between 3 and 7 hm (Fig. 3b). ATP content decreased between 0 and 3 hm in both MF and SF oocytes (Fig. 4a, b) but only in light-atretic MF oocytes, the decrease was significant (p < 0.05). On the other hand, in healthy MF oocytes, a significant increase in ATP content (p < 0.05) was found between 7 and 24 hm (Fig. 4 a). In SF oocytes, both healthy and light-atretic showed an increase in ATP content between 7 and 16 hm (Fig. 4b).
Discussion At present, there is not enough information about the relation of mitochondrial status to meiotic and
(a)
(b)
Fig. 2. Changes of mitochondrial activity in healthy and light-atretic MF (a) and SF (b) oocytes during maturation. Mean mitochondrial activity per oocyte was compared in the same category. Values with different superscripts within the same category of oocytes (A–Bin healthy; a–bin light-atretic) are significantly different (p < 0.05)
developmental competence in bovine oocytes. Some data indicate that mitochondrial reorganization and ATP levels differ between morphologically good and poor bovine oocytes and that these differences could be responsible for varying developmental capacity of embryos (Stojkovic et al. 2001; Tarazona et al. 2006). Correlations between the developmental potential and mitochondrial functional integrity have also been found in human, porcine and murine oocytes and embryos (Cummins 2004; Brevini et al. 2005; Nagai et al. 2006; Egerszegi et al. 2010). © 2014 Blackwell Verlag GmbH
Kinetics of the Mitochondrial Pattern in Bovine Oocytes
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(a)
(a)
(b)
(b)
Fig. 3. Changes of frequency of oocytes with mitochondrial clusters in healthy and light-atretic MF (a) and SF (b) oocytes during maturation. Frequency of oocytes with clusters was calculated in the same category. Values with different superscripts within the same category of oocytes (A–Cin healthy; a–cin light-atretic) are significantly different (p < 0.05)
Fig. 4. Changes of ATP content in healthy and light-atretic MF (a) and SF (b) oocytes during maturation. Mean ATP content per oocyte was compared in the same category. Values with different superscripts within the same category of oocytes (A–Cin healthy; a–cin light-atretic) are significantly different (p < 0.05)
In our preliminary study, we assessed that the mitochondrial status of bovine oocytes varies in meiotic competence before and after their maturation. To characterize mitochondria more objectively, we studied oocytes obtained from medium and small follicles separately and categorized them as healthy, light-atretic and mid-atretic (Machatkova et al. 2012). The study showed that, independently of meiotic competence, the values of mitochondrial parameters were significantly reduced in mid-atretic oocytes, as compared with those in light-atretic and healthy oocytes. The findings that a higher level of atresia negatively influenced mitochondrial patterns in bovine oocytes have also been reported by Stojkovic et al. (2001) and Tarazona et al. (2006). Therefore, mid-atretic oocytes were eliminated from the experiments of the present study, and only healthy and light-atretic oocytes, which are usually used for IVM and IVF, were included. This study was designed to assess the mitochondrial changes in healthy and light-atretic bovine oocytes with greater meiotic competence from medium follicles (MF) or lesser meiotic competence from small follicles (SF) during maturation in more detail, related to the resumption of meiosis, MII onset and completion of maturation, because this information is still missing.
Regarding the resumption of meiosis in oocytes, it has been reported that GVBD progresses faster in oocytes with greater meiotic competence than in those with lesser meiotic competence and that nuclear maturation is affected by follicular atresia (Liu et al. 2006). In this study, the resumption of meiosis was accelerated in light-atretic oocytes compared with healthy oocytes regardless of meiotic competence. This is in agreement with the opinion that a low level of atresia improves oocyte competence via its premature effect (Hendriksen et al. 2000). As light-atretic oocytes are subjected to the follicular microenvironment for a longer time, they are more differentiated than healthy oocytes (Blondin and Sirard 1995; Sirard et al. 1999, 2006). In our study, despite of an accelerated MII onset, the ability to complete nuclear maturation was reduced in lightatretic less competent oocytes in comparison with the other oocyte categories. Our explanation may be based on the view of Lodde et al. (2007) that, in atretic oocytes, interruption of communication between cumulus cells and oocytes can occur before nuclear maturation is completed. An increase in mitochondrial activity after oocyte maturation has been described in bovine (Tarazona
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M Jeseta, D Ctvrtlikova Knitlova, K Hanzalova, P Hulinska, S Hanulakova, I Milakovic, L Nemcova, J Kanka and M Machatkova
et al. 2006), porcine (Torner et al. 2004), hamster (Suzuki et al. 2005) as well as human oocytes (Wilding et al. 2001). However, no data have been reported on changes in mitochondrial patterns during maturation of bovine oocyte. Our study is the first to describe the course of mitochondrial changes during maturation of bovine oocytes varying in meiotic competence. This study confirmed that changes in mitochondrial activity during the course of maturation were related to meiotic competence of oocytes. Healthy more competent oocytes significantly increased their mitochondrial activity twice, before resumption of meiosis and before completion of maturation, while healthy less competent oocytes increased their mitochondrial activity significantly only once, before completion of maturation. Atresia influenced the course of changes in mitochondrial activity in both more and less competent oocytes. In light-atretic more competent oocytes, a decrease in mitochondrial activity from GV stage to resumption of meiosis and a subsequent increase until completion of maturation were observed compared with healthy more competent oocytes. On the other hand, light-atretic less competent oocytes were increasing their mitochondrial activity gradually until the MII onset reversely to healthy less competent oocytes. The heterogeneous distribution of mitochondria has been described as a marker of the activation of processes necessary for cytoplasmic maturation of oocytes (Torner et al. 2004; Van Blerkom 2004). It is also known that the reorganization of mitochondria is associated with ATP production (Yu et al. 2010). Our results confirmed a relationship between mitochondrial cluster formation and ATP production, which has also been found by other authors in bovine, human and murine oocytes (Stojkovic et al. 2001; Van Blerkom 2004; Yu et al. 2010). In addition, our study showed that mitochondrial reorganization and ATP production were dependent on both the meiotic competence and health of maturing oocytes. Healthy more competent
References Bavister BD, Squirrell JM, 2000: Mitochondrial distribution and function in oocytes and early embryos. Hum Reprod 15, 189–198. Blondin P, Sirard MA, 1995: Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes. Mol Reprod Dev 41, 54–62. Brevini TA, Vassena R, Francisci C, Gandolfi F, 2005: Role of adenosine triphosphate, active mitochondria, and microtubules in the acquisition of developmental competence of parthenogenetically activated pig oocytes. Biol Reprod 72, 1218–1223. Brevini TA, Cillo F, Antonini S, Gandolfi F, 2007: Cytoplasmic remodelling and the acquisition of developmental competence in pig oocytes. Animal Reprod Sci 98, 23– 38. Cao X, Chen Y, 2009: Mitochondria and calcium signaling in embryonic development. Sem Cell Dev Biol 230, 337–345.
oocytes significantly increased cluster formation and ATP production for the whole time of maturation, from meiosis resumption, until maturation was completed. On the other hand, light-atretic more competent oocytes and healthy less competent oocytes reduced these activities earlier, before maturation was completed. An interesting finding was that light-atretic less competent oocytes were the first to significantly increase cluster formation, between the GV stage and meiotic resumption. This fact correlates with the observed acceleration of GVBD in these oocytes. We assume that less competent light-atretic oocytes needed to replenish ATP stock to initiate the GVBD process. In conclusion, during maturation, there are specific differences in chromatin and mitochondrial patterns among bovine oocytes with different meiotic competences. The kinetics of mitochondrial changes is influenced not only by an inherent competence of the oocytes but also by their health. This finding can contribute to an understanding of processes associated with nuclear and cytoplasmic maturation of oocytes and to a supplementation of maturation medium by mitochondrial stimulants to improve embryonal development and in vitro embryo production. Acknowledgement This study was supported by Grants QI 91A018 and 0002716202 of the Ministry of Agriculture of the Czech Republic.
Conflict of interest None of the authors have any conflict of interest to declare.
Author contributions Marie Machatkova, Michal Jeseta, Drahomira Ctvrtlikova Knitlova designed study, analysed data and drafted paper. Michal Jeseta, Drahomıra Ctvrtlikova Knitlova, Jiri Kanka, Pavlin Hulinska, Sarka Hanulakova, Lucie Nemcova, Katerina Hanzalova and Irena Milakovic performed the experiments and analysed the data.
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Submitted: 14 Nov 2013; Accepted: 8 Mar 2014 Author’s address (for correspondence): Michal Jeseta, Department of Genetics and Reproduction, Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic. E-mail: [email protected]
Mitochondrial Patterns in Bovine Oocytes with Different Meiotic Competence Related to Their in vitro Maturation M Jeseta1, D Ctvrtlikova Knitlova1, K Hanzalova1, P Hulinska1, S Hanulakova1, I Milakovic1, L Nemcova2, J Kanka2 and M Machatkova1 1 Veterinary Research Institute, Brno, Czech Republic; 2Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Libechov, Czech Republic
Contents This study was designed to specify chromatin and mitochondrial patterns in bovine oocytes with different meiotic competence in relation to maturation progress, resumption of meiosis, MII onset and completion of maturation. Oocytes with greater or lesser meiotic competence, recovered separately from medium (MF) and small follicles (SF), were categorized according to morphology. Four oocyte categories, healthy and light-atretic MF and healthy and light-atretic SF oocytes were matured and collected at 0, 3, 7, 16 and 24 h of maturation. Specific differences in terms of chromatin and mitochondrial patterns were found among the maturing oocyte categories. Resumption of meiosis was accelerated in light-atretic oocytes, as compared with healthy oocytes, regardless of their meiotic competence. More competent oocytes activated mitochondria twice during maturation, before resumption of meiosis and before completion of maturation, while less competent oocytes did it only once, before completion of maturation. Changes in mitochondrial activity differed in light-atretic compared with healthy in both more and less competent oocytes. Healthy meiotically more competent oocytes formed clusters and produced ATP for the whole time of maturation until its completion, while light-atretic more competent oocytes and healthy less competent oocytes reduced these activities earlier, at MII onset. Contrary to these oocyte categories, light-atretic less competent oocytes increased cluster formation significantly before resumption of meiosis. It can be concluded that bovine oocytes with different meiotic competence and health differed in the kinetics of mitochondrial patterns during maturation.
Introduction At the present time, the developmental competence of bovine oocytes is a limiting factor for wider application of assisted reproductive technologies in cattle breeding. Even though maturation is close to optimum and fertilization is effective, development of fertilized oocytes to transferable embryos is not adequate. To improve embryo development, enough knowledge of the intrinsic quality of both immature and mature oocytes is required. It is generally known that the developmental ability of an embryo is determined by an initial potential of the oocyte, which reflects follicular conditions at the time of its recovery. In this context, the meiotic and developmental competence of the oocyte can be expressed by both size and health of the follicle, and its status can be assessed on the basis of cumulus– oocyte morphology (Sirard and Blondin 1996). Oocytes with the first signs of atresia and a less compact cumulus originating from larger or early-atretic follicles are more developmentally competent than those from smaller or © 2014 Blackwell Verlag GmbH
late-atretic follicles (Blondin and Sirard 1995; Sirard and Blondin 1996; Feng et al. 2007). Oocyte maturation is accompanied by major changes in the ooplasm, which lead to the completion of nuclear and cytoplasmic maturation (Brevini et al. 2007). In both processes, as well as in fertilization and early embryo development, mitochondria play an important role in matching adenosine triphosphate (ATP) supply to demand (Bavister and Squirrell 2000; Sathananthan and Trounson 2000; Brevini et al. 2005; Tarazona et al. 2006; Van Blerkom and Davis 2007; Van Blerkom 2008). Mitochondria in oocytes and embryos are important not only for cellular ATP production but also other functions, such as participation in calcium and redox signalling (Van Blerkom 2011). They regulate cellular calcium homeostasis and ROS defence which are critical to cell survival and embryonic development (Dumollard et al. 2006; Cao and Chen 2009). Mitochondrial dysfunction with low ATP production is one of the major factors compromising oocyte quality (Dumollard et al. 2006; Yu et al. 2010). Mitochondria are also known to be involved in oocyte atresia (Torner et al. 2004; Wang et al. 2009). The leakage of cytochrome C from dying mitochondria is a key event in the cascade leading to apoptosis (Shimizu et al. 1999). For these reasons, mitochondrial patterns can be taken as markers of the quality and developmental capacity of mammalian oocytes and embryos (Nagai et al. 2006; Egerszegi et al. 2010). However, two aspects are important for assessment of mitochondria: their quality and quantity (Cummins 2004). The morphology of mitochondria (Van Blerkom 2004), their aggregation (Torner et al. 2004) and gene expression (Kanka et al. 2012) have been described in relation to the developmental potential of oocytes. Mitochondrial aggregates with the smooth endoplasmic reticulum (SER), which are probably involved in Ca2+ release from SER and subsequent energy production, have been found in human oocytes matured in vivo (Van Blerkom 2011). Mitochondrial clusters are usually used as a criterion of cytoplasmic quality in oocytes matured in vitro (KatskaKsiazkiewicz et al. 2011). Although the importance of mitochondria for oocyte development is known and is likely to increase in the future (Wang et al. 2009), there is still a lack of information about their functional changes during oocyte maturation. In our previous study, we concluded that, before maturation, meiotically more competent oocytes had
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M Jeseta, D Ctvrtlikova Knitlova, K Hanzalova, P Hulinska, S Hanulakova, I Milakovic, L Nemcova, J Kanka and M Machatkova
more active and reorganized mitochondria than meiotically less competent oocytes. Although the less competent oocytes showed an increase in mitochondrial activity after maturation, they remained inferior to more competent oocytes in both cluster formation and ATP production (Machatkova et al. 2012). This study was designed to characterize mitochondrial patterns in bovine oocytes with different meiotic competence in the course of maturation, related to the resumption of meiosis, MII onset and completion of maturation.
Materials and Methods All chemicals used in this study were purchased from Sigma-Aldrich Chemical Co. (Prague, Czech Republic) unless otherwise stated. Oocyte collection and categorization Slaughtered Holstein dairy cows (total no = 131), aged 4–6 years, with a checked ovarian cycle stage, were used in the experiments. Ovaries in growth/stagnation and regression phases of folliculogenesis, assessed by follicular and corpus luteum (CL) morphology, were used for oocyte collection. The oocytes were recovered separately from medium (MF; 6–10 mm) and small (SF; 2–5 mm) follicles by aspiration and slicing, respectively. The cumulus–oocyte complexes (COC) suitable for IVM and IVF were selected and categorized as healthy (homogenous cytoplasm without dark spots and compact homogenous cumuli) or light-atretic (slightly granulated cytoplasm with ≤ two dark spots and expanded outer layers of cumuli) according to the Blondin and Sirard classification (Blondin and Sirard 1995). Four oocyte categories, healthy MF, light-atretic MF, healthy SF and light-atretic SF, were used in the experiments. Oocyte maturation The four oocyte categories were matured separately in TCM-199 medium (Earle0 s salts), supplemented with 20 mM sodium pyruvate, 50 IU/ml penicillin, 50 lg/ml streptomycin, 5% oestrus cow serum (ECS; Sevapharma, Prague, Czech Republic) and gonadotropins (P.G. 600 15 IU/ml; Intervet, Boxmeer, Holland) in four-well (a)
plates (Nunclon Intermed, Roskilde, Denmark) at 38.8°C under 5% CO2 in air. An adequate part of oocytes of each category was recovered at 0, 3, 7, 16 and 24 h of maturation (hm), denuded of cumulus cells and examined. Oocyte staining Mitochondria were stained in PBS supplemented with 0.4% BSA and 200 nM MitoTracker Orange CMTM Ros dye (Molecular Probes, Eugene, OR, USA) for 30 min at 38.8°C. Only respiring mitochondria were stained with this cell-permeant mitochondrial-specific dye. After washing, the oocytes were fixed in 3.7% paraformaldehyde for 60 min at room temperature. They were washed in PBS and mounted on glass slides, avoiding oocyte compression, using Vectashield medium (Vector Lab, Burlingame, CA, USA) containing 1 lM of DNA dye (SYTOX Green; Invitrogen; Carlsbad, CA, USA) specific for dyeing of chromatin. The slides were stored below 0°C until examined. Oocyte examination The oocytes were examined with the use of a laser scanning confocal microscope (Leica TCS SP2 AOBS; Leica, Heidelberg, Germany) equipped with Ar and DPSS lasers. The 488-nm excitation band and 490- to 515-nm detector and the 561-nm excitation band and 565- to 600-nm detector were used for chromatin and mitochondria visualization, respectively. The 40 9 Leica HCX PL APO CS objective, pinhole, offsets, gain and AOBS were adjusted. These parameters were kept throughout the whole experiment. The oocytes were scanned in equatorial optical sections, and microphotographs were saved and processed using the NIS-ELEMENTS AR 3.0 software (Laboratory Imaging, Prague, Czech Republic). Chromatin and mitochondrial status assessment A total of 1180 oocytes were evaluated for nuclear maturation and mitochondrial status. The GV, GVBD and MII rates, mitochondrial activity, expressed as a mean fluorescence per oocyte, and a frequency of (b)
Fig. 1. Representative images of oocytes without (a) or with (b) mitochondrial clusters demonstrated by confocal microscopy. Oocyte was stained by MitoTracker Orange CMTM Ros. Scale bar represents 20 lm
© 2014 Blackwell Verlag GmbH
Kinetics of the Mitochondrial Pattern in Bovine Oocytes
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Tukey’s post hoc test using ANOVA SPSS version 11.5 for Windows (SPSS, Inc. Chicago, IL, USA).
Table 1. Nuclear maturation in the oocyte categories at different maturation intervals Hours of maturation (hm) 0
Oocytes MF Healthy Light-atretic SF Healthy Light-atretic
3
7
16
Chromatin configuration rate (%) GV GVBD MII
n
GV
194 160
100.0a 100.0a
97.6a 100.0a
26.8a 51.4b,c
13.3a 35.0b
412 414
100.0a 98.3a
100.0a 100.0a
46.4b 66.1c
19.4a,b 24.0b
Results
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Comparison of the oocyte categories The four oocyte categories were compared in terms of nuclear maturation, mitochondrial activity, mitochondrial cluster formation and ATP production in the selected maturation intervals (Tables 1–4). A comparison of chromatin configuration showed that, in MF oocytes, the GVBD rates at 7 hm and MII rates at 16 hm were significantly higher (p < 0.05) in light-atretic than in healthy oocytes. At 24 hm, however, the maximum MII rate was achieved by both healthy and light-atretic MF oocytes. In SF oocytes, a significantly higher GVBD rate at 7 hm and a higher MII rate at 16 hm were found in light-atretic oocytes, as compared with healthy oocytes. At 24 hm, the MII rate of light-atretic oocytes had the lowest value of all oocyte categories (Table 1). Mitochondrial activity was significantly higher in healthy and light-atretic MF and healthy SF oocytes up to 7 hm, as compared with light-atretic SF oocytes (p < 0.05). From this time on, there were no differences in mitochondrial activity among the four oocyte categories (Table 2). An increased frequency of oocytes with clusters was observed in light-atretic SF oocytes at 7 hm. At 24 hm,
MII
100.0a 100.0a 93.5a,b 84.6b
Values with different superscripts within the same column are significantly different (p < 0.05).
oocytes with mitochondrial clusters were evaluated in the four oocyte categories. The morphology of oocytes with the presence or absence of clusters is shown in Fig. 1a,b, respectively. ATP content assessment A total of 210 oocytes were examined for ATP content using a bioluminescent somatic cell assay kit (FLASC). Briefly, the oocytes were rinsed three times in Ca2+- and Mg2+-free PBS and transferred individually, in 50 ll of solution, into plastic tubes. After adding 100 ll ice-cold somatic cell reagent (FLSAR) to each tube, the oocytes were incubated on ice for 5 min. Then, 100 ll ice-cold assay mix (FLAAM assay mix and FLAAB dilution buffer; 1:25) was added to each tube. The tubes were kept for an additional 5 min at room temperature in darkness. The intensity of luminescence (luciferin–luciferase reaction) was measured using Luminoskan (type 391A; Labsystems, Helsinki, Finland). A nine-point standard curve was determined, and a negative control was run for every 20 oocytes. The ATP content per oocyte was calculated by the formula derived from a linear regression of the standard curve in the four oocyte categories.
Table 3. Frequency of oocytes with mitochondrial clusters in the oocyte categories at different maturation intervals Frequency of oocytes with clusters (%) Hours of maturation (hm) Oocytes MF Healthy Light-atretic SF Healthy Light-atretic
Statistical analysis At least four replicates were carried out for each oocyte category and each time interval. The results were analysed with the chi-square test, Student’s t-test and
n
0
3
7
16
24
190 157
0.0 0.0
14.63a 13.63a
17.07a,b 14.29a
29.27a 26.32a
57.50a 35.48b
310 330
0.0 0.0
16.67a 12.96a
19.60a,b 25.93b
32.80a 29.94a
41.70b 43.08b
Values with different superscripts within the same column are significantly different (p < 0.05).
Table 2. Mitochondrial activity in the oocyte categories at different maturation intervals Mitochondrial activity per oocyte (mean SD) Hours of maturation (hm) Oocytes MF Healthy Light-atretic SF Healthy Light-atretic
n
0
3
7
16
24
166 148
30.1 11.2a 25.8 13.1a
22.3 13.5a,b 21.2 18.0a,b
42.6 12.8a 32.3 15.0a,b
16.4 9.9a 27.7 14.7a
29.9 22.4a 32.2 15.0a
257 307
28.4 21.2a 8.5 3.4b
30.8 13.8a 8.3 5.8b
29.4 11.8a,b 15.0 9.1b
21.1 14.6a 21.2 17.0a
36.7 20.8a 36.8 15.1a
Values with different superscripts within the same column are significantly different (p < 0.05).
© 2014 Blackwell Verlag GmbH
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M Jeseta, D Ctvrtlikova Knitlova, K Hanzalova, P Hulinska, S Hanulakova, I Milakovic, L Nemcova, J Kanka and M Machatkova
Table 4. ATP content in the oocyte categories at different maturation intervals ATP content per oocyte in pmol (mean SD) Hours of maturation (hm) Oocytes MF Healthy Light-atretic SF Healthy Light-atretic
n
0
3
7
16
24
115 103
2.70 0.48a 2.52 0.59a
2.25 0.24a 1.64 0.46a
2.33 0.36a 2.22 0.33a
2.71 0.29a 2.61 0.28a
3.30 0.34a 2.79 0.35a
296 279
2.31 0.45a 2.38 0.53a
2.13 0.35a 1.97 0.39a
2.10 0.31a 2.22 0.35a
2.76 0.33a 2.67 0.48a
3.01 0.39a 2.84 0.43a
Values with the same superscripts within the same column are not significantly different.
a significantly higher frequency of oocytes with clusters (p < 0.05) was found in healthy MF oocytes than in the other three categories (Table 3). No significant differences in ATP content were observed among the oocyte categories in any maturation interval (Table 4). Course of changes in the oocyte categories To characterize mitochondrial pattern during maturation, the changes in mitochondrial activity, frequency of oocytes with clusters and ATP content were expressed as curves, and a comparison of values for maturation times in each oocyte category was made. The results are presented in Figs 2–4. Mitochondrial activity changes during maturation differed between MF and SF oocytes (Fig. 2a,b). In healthy MF oocytes, there was a significant increase (p < 0.05) from 3 to 7 hm and a significant decrease from 7 to 16 hm. In light-atretic MF oocytes, the curve was affected by atresia (Fig. 2a). In SF oocytes, a significant increase in mitochondrial activity in both healthy and light-atretic oocytes was found only from 16 to 24 hm (Fig. 2b). The frequency of oocytes with clusters increased in both healthy and light-atretic MF and SF oocytes (Fig. 3a,b). In healthy MF oocytes, a significant increase (p < 0.05) was observed from 7 to 16 hm and from 16 to 24 hm, while, in light-atretic MF oocytes, it was only between 7 and 16 hm (Fig. 3a). In healthy SF oocytes, a significant increase (p < 0.05) was observed from 7 to 16 hm. On the other hand, in light-atretic SF oocytes, the frequency of oocytes with clusters significantly increased earlier, between 3 and 7 hm (Fig. 3b). ATP content decreased between 0 and 3 hm in both MF and SF oocytes (Fig. 4a, b) but only in light-atretic MF oocytes, the decrease was significant (p < 0.05). On the other hand, in healthy MF oocytes, a significant increase in ATP content (p < 0.05) was found between 7 and 24 hm (Fig. 4 a). In SF oocytes, both healthy and light-atretic showed an increase in ATP content between 7 and 16 hm (Fig. 4b).
Discussion At present, there is not enough information about the relation of mitochondrial status to meiotic and
(a)
(b)
Fig. 2. Changes of mitochondrial activity in healthy and light-atretic MF (a) and SF (b) oocytes during maturation. Mean mitochondrial activity per oocyte was compared in the same category. Values with different superscripts within the same category of oocytes (A–Bin healthy; a–bin light-atretic) are significantly different (p < 0.05)
developmental competence in bovine oocytes. Some data indicate that mitochondrial reorganization and ATP levels differ between morphologically good and poor bovine oocytes and that these differences could be responsible for varying developmental capacity of embryos (Stojkovic et al. 2001; Tarazona et al. 2006). Correlations between the developmental potential and mitochondrial functional integrity have also been found in human, porcine and murine oocytes and embryos (Cummins 2004; Brevini et al. 2005; Nagai et al. 2006; Egerszegi et al. 2010). © 2014 Blackwell Verlag GmbH
Kinetics of the Mitochondrial Pattern in Bovine Oocytes
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(a)
(a)
(b)
(b)
Fig. 3. Changes of frequency of oocytes with mitochondrial clusters in healthy and light-atretic MF (a) and SF (b) oocytes during maturation. Frequency of oocytes with clusters was calculated in the same category. Values with different superscripts within the same category of oocytes (A–Cin healthy; a–cin light-atretic) are significantly different (p < 0.05)
Fig. 4. Changes of ATP content in healthy and light-atretic MF (a) and SF (b) oocytes during maturation. Mean ATP content per oocyte was compared in the same category. Values with different superscripts within the same category of oocytes (A–Cin healthy; a–cin light-atretic) are significantly different (p < 0.05)
In our preliminary study, we assessed that the mitochondrial status of bovine oocytes varies in meiotic competence before and after their maturation. To characterize mitochondria more objectively, we studied oocytes obtained from medium and small follicles separately and categorized them as healthy, light-atretic and mid-atretic (Machatkova et al. 2012). The study showed that, independently of meiotic competence, the values of mitochondrial parameters were significantly reduced in mid-atretic oocytes, as compared with those in light-atretic and healthy oocytes. The findings that a higher level of atresia negatively influenced mitochondrial patterns in bovine oocytes have also been reported by Stojkovic et al. (2001) and Tarazona et al. (2006). Therefore, mid-atretic oocytes were eliminated from the experiments of the present study, and only healthy and light-atretic oocytes, which are usually used for IVM and IVF, were included. This study was designed to assess the mitochondrial changes in healthy and light-atretic bovine oocytes with greater meiotic competence from medium follicles (MF) or lesser meiotic competence from small follicles (SF) during maturation in more detail, related to the resumption of meiosis, MII onset and completion of maturation, because this information is still missing.
Regarding the resumption of meiosis in oocytes, it has been reported that GVBD progresses faster in oocytes with greater meiotic competence than in those with lesser meiotic competence and that nuclear maturation is affected by follicular atresia (Liu et al. 2006). In this study, the resumption of meiosis was accelerated in light-atretic oocytes compared with healthy oocytes regardless of meiotic competence. This is in agreement with the opinion that a low level of atresia improves oocyte competence via its premature effect (Hendriksen et al. 2000). As light-atretic oocytes are subjected to the follicular microenvironment for a longer time, they are more differentiated than healthy oocytes (Blondin and Sirard 1995; Sirard et al. 1999, 2006). In our study, despite of an accelerated MII onset, the ability to complete nuclear maturation was reduced in lightatretic less competent oocytes in comparison with the other oocyte categories. Our explanation may be based on the view of Lodde et al. (2007) that, in atretic oocytes, interruption of communication between cumulus cells and oocytes can occur before nuclear maturation is completed. An increase in mitochondrial activity after oocyte maturation has been described in bovine (Tarazona
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M Jeseta, D Ctvrtlikova Knitlova, K Hanzalova, P Hulinska, S Hanulakova, I Milakovic, L Nemcova, J Kanka and M Machatkova
et al. 2006), porcine (Torner et al. 2004), hamster (Suzuki et al. 2005) as well as human oocytes (Wilding et al. 2001). However, no data have been reported on changes in mitochondrial patterns during maturation of bovine oocyte. Our study is the first to describe the course of mitochondrial changes during maturation of bovine oocytes varying in meiotic competence. This study confirmed that changes in mitochondrial activity during the course of maturation were related to meiotic competence of oocytes. Healthy more competent oocytes significantly increased their mitochondrial activity twice, before resumption of meiosis and before completion of maturation, while healthy less competent oocytes increased their mitochondrial activity significantly only once, before completion of maturation. Atresia influenced the course of changes in mitochondrial activity in both more and less competent oocytes. In light-atretic more competent oocytes, a decrease in mitochondrial activity from GV stage to resumption of meiosis and a subsequent increase until completion of maturation were observed compared with healthy more competent oocytes. On the other hand, light-atretic less competent oocytes were increasing their mitochondrial activity gradually until the MII onset reversely to healthy less competent oocytes. The heterogeneous distribution of mitochondria has been described as a marker of the activation of processes necessary for cytoplasmic maturation of oocytes (Torner et al. 2004; Van Blerkom 2004). It is also known that the reorganization of mitochondria is associated with ATP production (Yu et al. 2010). Our results confirmed a relationship between mitochondrial cluster formation and ATP production, which has also been found by other authors in bovine, human and murine oocytes (Stojkovic et al. 2001; Van Blerkom 2004; Yu et al. 2010). In addition, our study showed that mitochondrial reorganization and ATP production were dependent on both the meiotic competence and health of maturing oocytes. Healthy more competent
References Bavister BD, Squirrell JM, 2000: Mitochondrial distribution and function in oocytes and early embryos. Hum Reprod 15, 189–198. Blondin P, Sirard MA, 1995: Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes. Mol Reprod Dev 41, 54–62. Brevini TA, Vassena R, Francisci C, Gandolfi F, 2005: Role of adenosine triphosphate, active mitochondria, and microtubules in the acquisition of developmental competence of parthenogenetically activated pig oocytes. Biol Reprod 72, 1218–1223. Brevini TA, Cillo F, Antonini S, Gandolfi F, 2007: Cytoplasmic remodelling and the acquisition of developmental competence in pig oocytes. Animal Reprod Sci 98, 23– 38. Cao X, Chen Y, 2009: Mitochondria and calcium signaling in embryonic development. Sem Cell Dev Biol 230, 337–345.
oocytes significantly increased cluster formation and ATP production for the whole time of maturation, from meiosis resumption, until maturation was completed. On the other hand, light-atretic more competent oocytes and healthy less competent oocytes reduced these activities earlier, before maturation was completed. An interesting finding was that light-atretic less competent oocytes were the first to significantly increase cluster formation, between the GV stage and meiotic resumption. This fact correlates with the observed acceleration of GVBD in these oocytes. We assume that less competent light-atretic oocytes needed to replenish ATP stock to initiate the GVBD process. In conclusion, during maturation, there are specific differences in chromatin and mitochondrial patterns among bovine oocytes with different meiotic competences. The kinetics of mitochondrial changes is influenced not only by an inherent competence of the oocytes but also by their health. This finding can contribute to an understanding of processes associated with nuclear and cytoplasmic maturation of oocytes and to a supplementation of maturation medium by mitochondrial stimulants to improve embryonal development and in vitro embryo production. Acknowledgement This study was supported by Grants QI 91A018 and 0002716202 of the Ministry of Agriculture of the Czech Republic.
Conflict of interest None of the authors have any conflict of interest to declare.
Author contributions Marie Machatkova, Michal Jeseta, Drahomira Ctvrtlikova Knitlova designed study, analysed data and drafted paper. Michal Jeseta, Drahomıra Ctvrtlikova Knitlova, Jiri Kanka, Pavlin Hulinska, Sarka Hanulakova, Lucie Nemcova, Katerina Hanzalova and Irena Milakovic performed the experiments and analysed the data.
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Submitted: 14 Nov 2013; Accepted: 8 Mar 2014 Author’s address (for correspondence): Michal Jeseta, Department of Genetics and Reproduction, Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic. E-mail: [email protected]
