Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation

Theriogenology 61 (2004) 1675–1689

Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation H. Tornera,*, K.-P. Bru¨ssowa, H. Alma, J. Ratkyb, R. Po¨hlanda, A. Tuchscherera, W. Kanitza a

Research Institute for the Biology of Farm Animals, 18196 Dummerstorf, Germany Department of Reproductive Biology, Research Institute for Animal Breeding and Nutrition, 2053 Herceghalom, Hungary

b

Received 28 April 2003; received in revised form 4 September 2003; accepted 11 September 2003

Abstract In this study, we evaluated the distribution and oxidative activity of mitochondria in ex vivo preovulatory porcine oocytes using the fluorescence probe MitoTracker CMTM Ros Orange. Cumulus– oocyte complexes (COCs) were classified according to cumulus morphology and time from hCG administration. The meiotic configuration of the oocytes and the degree of apoptosis in the surrounding cumulus cells were also evaluated. Estrus was synchronized in 45 crossbred Landrace gilts by feeding altrenogest for 15 days and administering 1000 IU PMSG on Day 16. The LH peak was simulated by treatment with 500 IU hCG, given 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before or 10, 22, or 34 h after hCG administration. Altogether 454 COCs were aspirated from follicles with a diameter of more than 5 mm. Cumulus morphology in the majority of COCs recovered 2 h before and 10 h after hCG was compact (60.4 and 52.7%, respectively; P < 0:05). At 22 h after hCG, COC morphology changed significantly from 10 h dramatically: 74% of COCs had an expanded cumulus (P < 0:01). At 34 h after hCG, 100% of recovered COCs had an expanded cumulus. The percentage of oocytes with a mature meiotic configuration differed among COC morphologies and increased as the interval after hCG administration increased (P < 0:05). The type of mitochondrial distribution in the oocytes (n ¼ 336) changed from homogeneous to heterogeneous as the interval after hCG administration increased (P < 0:01) and was associated with the cumulus morphology. Representative mitochondrial distributions were found as follows: 2 h: fine homogeneous in compact and dispersed COCs; 10 h: granulated homogeneous in compact and * Corresponding author. Tel.: þ49-38208-68759; fax: þ49-38208-68752. E-mail address: [email protected] (H. Torner).

0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.09.013

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dispersed COCs; 22 h: granulated homogeneous in expanded COCs; and 34 h: granulated heterogeneous and clustered heterogeneous in expanded COCs (P < 0:01). The oxidative activity of mitochondria measured by fluorescence intensity (Em: 570 nm) per oocyte after Mitotracker CMTM Ros Orange labeling increased in the oocyte as the post-hCG interval increased (P < 0:01) and depended on the type of mitochondrial distribution. Lowest oxidative activity of mitochondria was found in oocytes with fine homogeneous distribution (253:1  9:4 mA). The oxidative activity increased (334:4  10:3 mA) in oocytes with granulated homogeneous distribution of mitochondria, and reached highest level in oocytes with granulated heterogeneous (400:9  13:0 mA) and clustered heterogeneous distributions (492:8  13:9 mA) (P < 0:01). Mitochondrial activity in oocytes coincided with apoptosis in surrounding cumulus cells which increased in a time-dependent manner during pre-ovulatory maturation in vivo (P < 0:01). These results indicate that there is a relationship between meiotic progression, cumulus expansion and mitochondrial redistribution and their oxidative activity during final pre-ovulatory maturation in pig oocytes. It appears that increased levels of mitochondrial activities in oocytes are correlated to increased levels of apoptosis in surrounding cumulus cells, in which mitochondria may play a role. # 2003 Elsevier Inc. All rights reserved. Keywords: Mitochondrial activity; Apoptosis; Porcine oocytes; Pre-ovulatory maturation

1. Introduction The application of technology for in vitro maturation and in vitro fertilization (IVM/ IVF) in pigs depends upon establishment of techniques for repeatable oocyte recovery and on knowledge of oocyte maturation during pre-ovulatory follicular development. There is little information available regarding the changes in the cumulus, the oocyte cytoplasm, and the oocyte nucleus during final follicular maturation in vivo in the pig. Mitochondria play a vital role in the oocyte to provide ATP for fertilization and preimplantation embryo development. Data in human and bovine oocytes suggest that the efficiency of mitochondrial respiration in oocytes is closely correlated with the rate of embryo development after fertilization [1,2]. Electron microscopy studies of oocytes have revealed dynamic morphological changes of mitochondria during the pre-ovulatory period. At this time, they are the most prominent organelles in the ooplasm [3]. They form voluminous aggregates with the smooth endoplasmatic reticulum (SER), tubules and vesicles. These mitochondrial-SER aggregates (m-SER) could be involved in the production of a reservoir of energy prior to fertilization. However, the role of mitochondria during maturation, fertilization, and embryonic development is not fully understood [4]. There appear to be large differences in the timing of changes in activity and in distribution of mitochondria during oocyte maturation among species (sheep [5]; human [1], mouse [6,7], cattle [2], sea urchin [8]). For the most part, these studies have been done in vitro. Sun et al. [9] used the fluorescence probe MitoTracker Green to stain mitochondria in IVM pig oocytes. This stain is not dependent upon membrane potential. They concluded that oocyte maturation in vitro was associated with changes in distribution of active mitochondria, and that in vitro conditions may cause incomplete movement of mitochondria to the inner cytoplasm and thus may affect cytoplasmic maturation.

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Mitochondria play a role in aging, cellular apoptosis, metabolism, and also in many diseases. They are controlled by a dual genome system, with cooperation between endogeneous mitochondrial genes and mitochondrial genes translocated to the nucleus over the course of evolution [10]. Mitochondria contain several pro-apoptotic molecules that activate cytosolic proteins to execute apoptosis, block anti-apoptotic proteins in the cytosol and directly cleave nuclear DNA. For example, over-expression of cytochrome c during mitochondrial respiratory chain function enhances caspase activation and promotes cell death in response to apoptotic stimulation [11]. In pig approximately 55% of all antral follicles undergo degeneration (atresia); during 3 days before oestrus, this increases to 85%, thus only 15% of the follicles survive to ovulate [12]. In cattle and camels, cumulus cells of COCs from healthy follicles show remarkable apoptotic changes related to cumulus expansion during IVM [13,14]. Thus, during the final stage of pre-ovulatory maturation, mitochondrial activation in oocytes and apoptosis in surrounding cumulus cells are dominating events. There have been no reports on the precise timing of changes in oocyte mitochondrial activity and distribution in relation to nuclear maturation in pig, nor on apoptosis in cumulus cells during the pre-ovulatory period. The aim of the present study was to determine the influence of the pre-ovulatory maturation stage (in relation to hCG administration) on nuclear maturation, mitochondrial aggregation patterns and activities of oocytes, and cumulus morphology and degree of apoptosis.

2. Materials and methods 2.1. Oocyte collection A total of 45 crossbred Landrace gilts aged 8.5 months with a body mass of 120–125 kg were used. Gilts were synchronized by feeding 20 mg altrenogest (Regumate1 SerumWerk Bernburg, Germany) per animal daily for 15 days. Follicle growth was stimulated by administering 1000 IU i.m. equine chorionic gonadotrophin (eCG; Pregmagon1, Dessau, Germany) 24 h after the altrenogest-feeding (08.00 h). Final follicle maturation was stimulated by treatment with 500 IU hCG 80 h after eCG treatment. Endoscopic oocyte recovery was carried out 2 h before and 10, 22 or 34 h after hCG. Only macroscopically healthy follicles, well-vascularized and translucent and with a diameter of >5 mm were punctured, as described by Bru¨ ssow and Ra`tky [15]. 2.2. Evaluation of recovered cumulus–oocyte complexes Follicular fluids from different follicles of one ovary were pooled, and the morphology of the freshly recovered COCs was determined using an inverted microscope at 60 magnification. The COCs were classified according to morphology of cumuli as compact, dispersed, slightly expanded, with only corona radiata or expanded [16]. To evaluate the chromatin configuration and degree of cumulus cell apoptosis at the time of recovery, 25% of the COCs recovered at each time period were classified according to cumulus

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morphology, then the cumulus cells were mechanically removed from the oocytes by repeated pipetting and subsequent treatment with 3% sodium citrate. The cumulus cells were retained for detection of apoptosis. The denuded oocytes were then mounted on slides and fixed for >24 h in a mixture of acetic acid/alcohol/chloroform (3 : 6 : 1) before staining with 2% orcein in 60% acetic acid. The nuclear configuration of 118 oocytes was examined by phase contrast optics at 250–630 magnification. Based on their nucleus status the oocytes were classified as (1) immature—germinal vesicle (GV), (2) resumption of meiosis—GV breakdown, diakinesis, metaphase I to anaphase I, or (3) mature—telophase I and metaphase II as described for cattle [17], pig [16] and horses [18,19]. The percentage of the cumulus cells having apoptotic nuclei was estimated using an apoptosis kit (Roche Diagnostics GmbH, Mannheim, Germany). For determination of apoptosis, the cumulus cells were separated, dried on poly-L-lysine coated slides and fixed in 4% formaldehyde. After fixation the slides were stained with TUNEL reaction mixture and propidium iodide, embedded and subsequently evaluated by fluorescence microscopy [13]. 2.3. Fluorescence labeling and measurement of fluorescence intensity The remainder of the COCs (336) recovered at each time period were processed to evaluate mitochondrial patterns and activities. Immediately after classifying the cumulus morphology, COCs were incubated for 30 min in PBS containing 3% BSA and 200 nM MitoTracker Orange CMTM Ros (Molecular Probes, Oregon, USA) under culture conditions. The cell-permeant probe MitoTracker Orange-fluorescent tetramethylrosamine (M-7510) is readily sequestered only by actively respiring organelles dependent upon their oxidative activity. Then the cumulus cells were removed as described above, and the oocytes were washed three times in pre-warmed PBS without BSA. The oocytes were then fixed for 15 min at 37 8C using freshly prepared 2% paraformaldehyde in Hank’s balanced salt solution. The probe M-7510 contains a thiol-reactive chloromethyl moiety and can react with accessible thiol groups on peptides and proteins to form an aldehyde-fixable fluorescent conjugate, which is well-retained after cell fixation over a period of six weeks. After fixation the oocytes were washed three times in PBS, mounted on slides under cover slips and stored in the refrigerator prior to fluorescence microscopy evaluation. An epifluorescence microscope (Jenalumar, Carl Zeiss, Jena, Germany) was used for all experiments. Emission wavelengths were separated by a 540 nm dichroic mirror followed by further filtering through a 570 nm long pass filter (red emission). The mitochondrial distribution pattern of pig oocytes was characterized only by observation (up to 500 magnification) of the labeled mitochondria which were oxidative active. The distribution patterns were mainly classified as homogeneous (even distribution throughout the cytoplasm), or heterogeneous (uneven distribution within the cytoplasm). The fluorescence intensity (mA) was measured by the Nikon Photometry System P 100 (Nikon, Du¨ sseldorf, Germany). Microscope adjustments and photomultiplier settings were kept constant for all experiments. Oocytes were positioned in the plane of focus, and the area of measurement was adapted to the size of the oocyte. The data of emission intensity/ oocyte were reduced by compensation for the background fluorescence.

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2.4. Statistical analyses Statistical analysis was done with the SAS System for Windows (release 8.02). Elementary statistics and frequency tables were calculated with the help of the procedures of MEANS and FREQ in SAS/BASE software. Moreover, the data were analyzed with the GENMOD procedure of SAS/STAT software on the basis of a statistical model formulated as generalized linear model by using logistic models for binary data, and log-linear models for multinomial data. The GENMOD procedure fits generalized linear models, as defined by Nelder and Wedderburn [20]. The class of generalized linear models is an extension of traditional linear models that allows for the mean of a population to depend on a linear predictor through a non linear link function and the response probability distribution to be any member of an exponential family of distributions. A GENMOD procedure Type 3 analysis, consisting of specifying a model and computing likelihood ratio statistics for Type III contrasts for each term in the model, was done. The results displayed in the tables and figures are means  S:E:M: Test results having first kind risks with P < 0:05 were considered to be significant.

3. Results Altogether, 454 COCs were aspirated from the ovaries of 45 gilts. The majority of COCs (60.4, 52.7%, P < 0:05) aspirated from early follicles (2 h before and 10 h after hCG administration, respectively) had compact cumuli (Table 1). Concurrently, the portion of dispersed COCs decreased between 2 and 10 h (P < 0:05). The cumulus morphology in COCs recovered 10 h after hCG was heterogeneous with all types of cumulus morphologies seen except expanded. Starting at about 22 h after hCG, COC morphology changed significantly to the expanded type (74.0%; P < 0:01), and at 34 h after hCG this portion increased further (P < 0:01) to 100% expanded COCs. The highest percentage of meiotically immature oocytes (in the stage of GV) was found in compact COCs at 2 and 10 h after hCG administration (76.9 and 75.0%, respectively; Table 2). The proportion of immature oocytes in dispersed COCs decreased as the posthCG interval increased (P < 0:05). The proportion of oocytes resuming meiosis was highest in oocytes with dispersed, slightly expanded and corona radiata cumuli at 10 h after Table 1 COC morphology at different times before and after hCG injection (n ¼ 454) Time after hCG injection (h)

2 10 22 34

Number of COCs (n)

COC morphology (%) Compact

Dispersed

Corona radiata

Slightly expanded

Expanded

96 127 119 112

60.4 52.7 0 0

39.6a 17.3b 26.0b,c 0

0 15.7 0 0

0 14.3 0 0

0 0 74.0d 100.0e

(a:b, d:e) P < 0:01; (a:c) P < 0:05 (in columns).

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Table 2 Meiotic configuration as related to COC morphology at different times before and after hCG injection (n ¼ 118) Time after hCG injection (h)

COC morphology

Number of COCs (n)

Chromatin configuration (%) Immature (GV)

Resumption of meiosis (GVBD—A I)

2

Compact Dispersed

13 14

76.9 57.1a

23.1 42.9

0 0

10

Compact Dispersed Corona radiata Slightly expanded

16 11 10 9

75.0 36.4 20.0 22.2

25.0 63.6 70.0 66.8

0 0 10.0 11.0

22

Dispersed Expanded

12 15

16.7b 0

66.6 53.3c

16.7 46.7e

34

Expanded

18

16.7d

83.3f

0

Mature (T I/M II)

(a:b, c:d, e:f) P < 0:05 (in columns).

hCG. A significant proportion of oocytes in metaphase II was observed only in expanded COCs, and this proportion increased from 47.7 to 83.3% in 22 to 34 h post-hCG administration (P < 0:05). Cumuli from all COC types at different times after hCG administration were evaluated for presence of apoptosis (Fig. 1). Because there were so few cells in the corona-radiataonly group, this COC type could not be used. During pre-ovulatory maturation (2 to 22 h

Fig. 1. Apoptosis in cumulus cells from pig during pre-ovulatory maturation depending on cumulus morphology (a:b; c:d; a, b, c, d, e:f) P < 0:01.

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Table 3 Mitochondrial distribution as related to COC morphology at different times before and after hCG injection (n ¼ 336) Time after hCG injection (h)

COC morphology

Number of COCs (n)

Mitochondrial distribution (%) Homogeneous

Heterogeneous

Fine

Crystalline

Granulated

Crystalline

Granulated

Cluster

60.0 100.0

40.0 0

0 0

0 0

0 0

0 0

2

Compact Dispersed

10

Compact 51 Dispersed 11 Slightly ex- 9 panded Corona radiata10

29.4c 0 0

21.6a 0 0

49.0b 100.0 100.0

0 0 0

0 0 0

0 0 0

0

0

0

100.0

0

0

22

Dispersed Expanded

19 73

0 13.7f

47.4 0

34

Expanded

94

10.6h

0

45 24

0 60.3d 0

0 0

52.6 12.3g

0 13.7e

0

39.4j

50.0i

(a:b, d:e, d:f, d:g, h:i, h:j) P < 0:01; (b:c) P < 0:05 (in rows).

post hCG) the proportion of apoptotic cells in compact and dispersed cumulus types increased, and this was significantly higher in dispersed cumuli (P < 0:01). The highest proportion of apoptotic cells were found in expanded cumuli 22 and 34 h after hCG (P < 0:01). As a whole, the proportion of apoptotic cells increased as the interval from hCG administration increased, and was associated with COC type. In Table 3 the mitochondrial distribution in oocytes is shown related to COC morphology, at different times before and after hCG injection. The mitochondrial distribution pattern of pig oocytes was characterized by two main distribution features: labeled mitochondria were distributed evenly throughout the cytoplasm (homogeneous); labeled mitochondria were distributed unevenly within the cytoplasm (heterogeneous). These two main groups of mitochondrial distribution were divided further concerning the aggregation of mitochondria: small pixels of fluorescence intensity—fine; small linear spots of fluorescence intensity—crystalline; bigger areas of fluorescence with irregular shapes—granulated; and aggregates of bigger fluorescent areas—cluster. The fine aggregation type was found in homogeneously distributed oocytes only; the cluster aggregation type only in the heterogeneously distributed oocytes. Heterogeneously distributed mitochondria were observed only in oocytes which were recovered 22 or 34 h after hCG and in oocytes with corona radiata only at 10 h after hCG (P < 0:01). The homogeneous distribution type was found in each cumulus category except oocytes with corona radiata only, and was prominent in oocytes at 2 and 10 h after hCG. Independent of type of mitochondrial distribution, the aggregation of mitochondria increased as the interval after hCG administration increased. Representative mitochondrial aggregation and distribution patterns in oocytes were as follows: 2 h: fine homogeneous in compact and dispersed COCs (Fig. 2); 10 h: granulated homogeneous in compact and dispersed COCs (Fig. 3); 22 h: granulated homogeneous in expanded COCs; and 34 h: granulated (Fig. 4) and clustered (Fig. 5) (heterogeneous) in expanded COCs.

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Fig. 2. Fine homogeneous mitochondria distribution in oocytes from compact and dispersed COCs after 2 h of hCG administration (250, MitoTracker M 7510 staining).

Fig. 3. Granulated homogeneous mitochondria distribution in oocytes from compact and dispersed COCs after 10 h of hCG administration (250, MitoTracker M 7510 staining).

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Fig. 4. Granulated heterogeneous mitochondria distribution in oocytes from dispersed and expanded COCs after 22 h of hCG administration (250, MitoTracker M 7510 staining).

Fig. 5. Clustered heterogeneous mitochondria distribution in oocytes from expanded COCs after 34 h of hCG administration (250, MitoTracker M 7510 staining).

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Table 4 Mitochondrial activity as related to mitochondrial distribution and COC morphology at different times before and after hCG injection (n ¼ 336) COC morphology

Fluorescence intensity per oocyte  S.E.M. (mA) type of mitochondrial distribution Homogeneous

Heterogeneous

Fine

Crystalline

Granulated

Crystalline

Granulated

Clustered

2

Compact Dispersed

258.4  15.4 289.1  16.3

266.1  18.9 –

– –

– –

– –

– –

10

Compact Dispersed Slightly expanded Corona radiata

255.9  20.6 – – –

328.5  24.1 – – –

320.9  16.0 328.9  24.1 361.3  26.7 –

– – – 342.8  25.3

– – – –

– – – –

22

Dispersed Expanded

– 222.7  25.3d

280.8  26.7 –

– 326.5  12.1e,h

– –

348.2  25.3 416.4  26.7e

– 477.5  25.3e,g

34

Expanded

239.4  25.3a

–

–

–

438.1  13.2b

508.2  11.7c

Total

253.1  9.4I

291.8  13.5j

334.4  10.3k

342.8  25.3l

400.9  13.0m

492.8  13.9n

(a:b:c, d:e, g:h) P < 0:01; (n:i, j, k, l, m; k:i; k:m; l:i; m:i; m:j) P < 0:01 (in rows).

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Time after hCG injection (h)

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To confirm that the fluorescence intensity of the emission light from the fixed MitoTrackerlabeled oocytes was independent of the interval of storage, we measured the fluorescence intensity of 10 different oocytes in intervals of 24 h during 10 days. The measured fluorescence intensity per fixed oocyte was not influenced by storage. All investigated oocytes were recovered and further processed for fluorescence intensity measurement in four consecutive experiments during two months. All oocytes from the experiments were evaluated 2 days after the follicle aspiration as a whole. The data in Table 4 demonstrate that the respiratory activity in the oocytes as measured by fluorescence intensity for 570 nm emission/oocyte is associated with the type of mitochondrial distribution and aggregation (P < 0:01) and with the time since hCG injection (P ¼ 0:02). The lowest fluorescence intensity/oocyte was found in oocytes with fine homogeneous mitochondrial distribution (253:1  9:4 mA). The intensity of fluorescence increased in oocytes with granulated homogeneous distribution of mitochondria (334:4  10:3 mA) and reached the highest level in oocytes with granulated heterogeneous (400:9  13:0 mA) and clustered heterogeneous distributions (492:8  13:8 mA) (P < 0:01). The measured fluorescence intensity/oocyte was the lowest in compact and dispersed COCs at 2 h before hCG and highest in expanded COCs 34 h after hCG (P < 0:05).

4. Discussion The present study was conducted to monitor the changes in mitochondria and chromatin of pig oocytes and of the apoptotic level in surrounding cumulus cells during pre-ovulatory maturation in vivo. MitoTracker Orange labeling of respiring mitochondria and photometric measurement of fluorescence intensity were used to determine the respiratory activity/oocyte. Our investigation shows, as in previous studies [16], that the oocytes progressed to metaphase II between 22 and 34 h after hCG administration. Similar results on meiotic progression of in vivo matured oocytes were obtained by Xie et al. [21]. The data show (Table 2) that the stage of meiotic progression is related both to the increased time interval after hCG administration and to changes in cumulus investment such as cumulus expansion. Most oocytes with a compact cumulus were in the GV-stage and most oocytes with expanded cumuli were in telophase I or metaphase II, indicating that cumulus cell expansion is associated with nuclear maturation. Porcine oocytes with expanded cumulus showed different degrees of nuclear maturation according to the time after hCG administration, indicating that cumulus expansion precedes nuclear maturation. Previous studies on the distribution of polysomal and free mRNA fractions in porcine cumulus cells found that the cumulus cells became translationally active at later stages of pre-ovulatory maturation (22 and 34 h post hCG) [22]. This is in accordance with our results in cumulus expansion and nuclear maturation in oocytes 22 h after hCG. Only cumuli of expanded COCs 22 h after hCG become translationally active with increasing level of activity depending on meiotic stage. Our results on the change in the cumulus morphology 22 h after hCG are similar to those reported by Cran [23]. All alterations in the oocyte nucleus and the surrounding cumulus of the observed porcine COCs were associated with dramatic changes in the respiratory activity, and the

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pattern of aggregation and distribution of ooplasmic mitochondria. During this short time of pre-ovulatory maturation (2 to 34 h post hCG) we observed a strong time-dependent mitochondrial aggregation and distribution (P < 0:05), and a time-dependent mitochondrial distribution patterns (P ¼ 0:02) on mitochondrial activity/oocyte (P < 0:01). Active mitochondria reorganization occurs, also during maturation in vitro in bovine oocytes [2]. Therefore, examination of these alterations in mitochondrial organization might serve as predictor of oocyte or embryo developmental competence. Mitochondria play a vital role in the metabolism of energy-dependent mechanism in the oocyte cytoplasm to provide ATP for fertilization and pre-implantation embryo development (1). In vitro maturation of bovine oocytes resulted in a significant increase in the average ATP content/oocyte (2). Because mitochondrial reorganization and ATP levels in this study were different between morphologically good and poor oocytes, the authors concluded that mitochondrial redistribution and activity in the oocytes may be responsible for their different developmental capacity after IVF [2]. Other studies in human oocytes [1], sheep oocytes [24], and rodent oocytes [4] support this hypothesis. In our experiment, two major mitochondrial distribution patterns were visualized. The first pattern, homogeneous distribution of mitochondria throughout the cytoplasm, was more common in oocytes at early stages of pre-ovulatory maturation (2 h before hCG, 10 h post hCG). The second pattern, heterogeneous distribution of mitochondria, was seen more often in oocytes in advanced stages of meiosis and having an expanded cumulus at later times after hCG administration (22 and 34 h post hCG). For each mitochondrial distribution type, there was a shift from the fine and crystalline subtype to the more aggregated granulated and clustered subtypes over time. The increased aggregation of mitochondria is clearly correlated to the increased post-hCG time interval. The distribution and location of ooplasmic mitochondria between species show subtle differences, so the comparison of mitochondrial function and physical location of the mitochondria are complicated [4]. Thus, a comparison between our findings (distribution types) and those of others is difficult, because either other species or in vitro studies were used. Nevertheless, our data is in accordance with studies in different species [2,3,9] in that we also observed an increased level of mitochondrial aggregation associated with oocyte maturation. Sathananthan and co-workers [25,26] reported that mitochondria in metaphase I and metaphase II human oocytes were numerous and evenly spread in the ooplasm, associating closely with vesicles or aggregates of tubular smooth endoplasmic reticulum. There are only few studies on mitochondrial activity in human, frog, and cattle oocytes during oogenesis [1,2,27]. No changes in the mitochondrial activity were noted during human oocyte maturation [1]. However, our findings are in accordance with those of other species, in that we found significant differences in the respiratory activity/oocyte between oocyte groups, depending on the type of mitochondrial distribution and aggregation, and on the post-hCG time interval administration. The mitochondrial activity is associated with both meiotic progression and cumulus expansion. Shortly before ovulation the mitochondrial activity/oocyte reached the highest intensity, in order to provide ATP for fertilization and pre-implantation embryo development. This highest level of respiratory activity, found in ooplasmic mitochondria at 22 and 34 h post hCG, should be considered in the light of previous data reporting a high level of polysomal active fraction (translationally active mRNA) in expanded cumulus cells at this time [22] and the present results on apoptosis in

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the surrounding cumulus cells. All of these parameters, i.e. mitochondrial activity in the oocyte, quantity of polysomal mRNA fraction, and the apoptotic level in the cumulus cells are increasing. Similar results concerning apoptosis are available from in vitro studies both in camel [13] and cattle [14] which reported an increasing level of apoptotic cumulus cells during IVM. One explanation for the increase of apoptosis in the cumulus cells during preovulatory maturation could be the process of differentiation which is a prerequisite for cumulus expansion and detachment [28]. This differentiation is driven also by active protein synthesis, as indicated in porcine cumulus cells by increased levels of polysomal active fractions [22]. Mitochondrial changes may also be related to the degree of apoptosis. Apoptosis involves an early mitochondrial activation, which is followed by cytochrome c release and activation of caspases, the initiators of DNA fragmentation in the cells [5,29,30]. In Xenopus laevis oocytes, it could be demonstrated that isolated and purified mitochondria from mouse liver cells induced nuclear apoptosis [31]. In mammalian pre-ovulatory follicles first of all the granulosa cells but not oocytes undergo apoptosis during atresia [32]. It may be, that because of the high number of mitochondria in the oocyte (about one million; [33]), activation of the mitochondria in the oocyte could be the starting point of apoptosis, as a signal for cell differentiation in the surrounding cumulus cells. The intercellular contact based on intercellular bridges between cumulus cells and the ooplasm was clearly demonstrated in Raja oocytes [34], in human oocytes [35], and bovine oocytes [36]. These intercellular bridges also contain mitochondria. The presence of such intercellular bridges offers may be the possibility for transport of caspase activating molecules from the activated ooplasmic mitochondria to the surrounding cumulus cells. In conclusion, mitochondrial distribution and aggregation patterns in porcine oocytes are influenced by the stage of pre-ovulatory maturation, including meiotic progression and cumulus expansion. Mitochondrial activity increased during final pre-ovulatory maturation of oocytes, and mitochondrial distribution pattern changed from homogeneous to granular and clustered. Increased levels of respiratory activity in the oocytes were associated with increased levels of apoptotic cells in the surrounding cumulus cells, which might be caused by pro-apoptotic stimulation of oocyte mitochondria.

Acknowledgements The authors thank Dr. Katrin Hinrichs, Texas A&M University for the review of the manuscript.

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