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BOR - Papers in Press, published online ahead of print May 5, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028613
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BIOLOGY OF REPRODUCTION 71, 790–796 (2004)
DOI: 10.1095/biolreprod.104.028613
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Role of Fas-Mediated Apoptosis and Follicle-Stimulating Hormone on the Developmental Capacity of Bovine Cumulus Oocyte Complexes In Vitro1

Francisco J. Rubio Pomar3, Bernard A.J. Roelen2,3, Karin A. Slot4, Helena T.A. van Tol3, Ben Colenbrander3, and Katja J. Teerds5

Department of Farm Animal Health3 Department of Cell Biology and Biochemistry,4 Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Department of Animal Sciences,5 Human and Animal Physiology Group, Wageningen University, Wageningen, The Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Follicular atresia is believed to be largely regulated by apoptosis. To further understand how apoptosis can affect cumulus cells and oocytes we have evaluated the incidence and regulation of apoptosis affecting bovine cumulus oocyte complexes in vitro. Expression of components of the Fas signaling pathway was studied in both oocytes and cumulus cells by polymerase chain reaction after reverse transcription, immunoblotting, and indirect immunofluorescence. Furthermore, the Fas signaling pathway was activated in cumulus oocyte complexes with an agonistic anti-Fas antibody during in vitro maturation in the presence or absence of FSH. Viability and incidence of apoptosis in cumulus cells were evaluated by assessing membrane integrity and nuclear morphology. Oocyte nuclear maturation was also analyzed, as well as cleavage rates, blastocyst formation rates, and blastocyst quality, following in vitro fertilization. Fas mRNA and protein were expressed both in oocytes and cumulus cells. FasL protein was found in cumulus cells but could not be detected in oocytes, despite its mRNA expression. Both activation of the Fas pathway and presence of FSH during in vitro maturation increased the incidence of apoptosis in cumulus cells, affecting predominantly the middle and peripheral regions of the cumulus. The observed increase, however, had no effect on the developmental competence of the oocytes.

apoptosis, cumulus cells, embryo, in vitro fertilization, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the ovary, folliculogenesis results from a complex interplay between proliferation, differentiation, and atresia. Throughout each follicular wave, promotion of the dominant follicle and atresia of the subordinate follicles occurs [1, 2]. During early atresia in antral follicles, the cumulus cells and the oocyte remain apparently unaffected by the atretic changes, which are primarily manifested as apoptosis in the mural granulosa cells and, at a later stage, in the theca cells [3–5]. It has been suggested that apoptosis of granulosa cells is regulated by the Fas signaling pathway [6–8]. Fas is a cell surface receptor that, when activated by its ligand FasL, induces activation of a series of cysteine proteases known as caspases [9, 10]. Two classes can be distinguished in the caspase family: initiator caspases and effector caspases. The activation of an effector caspase (e.g., caspase-3 in granulosa cells) [3, 11] by initiator caspases ultimately results in nuclear fragmentation and cell death [12]. In the bovine ovary, Fas and FasL expression has been reported in theca and granulosa cells [3, 6, 7], with mRNA and protein levels being higher in atretic follicles than in healthy dominant follicles [7]. The presence of Fas has also been demonstrated in cumulus cells of various other species such as the pig [13], mouse [14], and human [15], although little is known about the role of the Fas system in the activation of apoptosis in these cells.

Cumulus cells play an important role in the nuclear and cytoplasmic changes that occur in the oocyte during in vitro maturation (IVM) [16]. Because the developmental competence of oocytes is compromised if they originate from heavily atretic follicles [17–19], it can be assumed that apoptosis of cumulus cells affects oocyte maturation, thus diminishing the developmental capacity of the oocyte. In general, human oocytes surrounded by a cumulus investment with a low level of apoptosis or in which apoptosis is absent, have a better capacity to develop into good quality embryos after in vitro fertilization (IVF) and in vitro culture (IVC) than oocytes surrounded by cumulus cells showing a significant rate of apoptosis [20, 21]. During IVM of selected bovine cumulus oocyte complexes (COCs), spontaneous apoptosis has been demonstrated to occur [22], but whether this affects the developmental capacity of the maturing oocytes is at present unknown.

FSH induces cumulus expansion when added to bovine COCs during IVM and increases the developmental potential of the oocytes in terms of IVF outcome [23, 24]. The presence of FSH prevents apoptosis of bovine granulosa cells in vitro [4], but whether this protective effect also occurs in cumulus cells is not known. Processes affecting oocytes during IVM, while compatible with apparently normal maturation, fertilization, and cleavage rates, can lead to abnormalities in embryo development such as DNA fragmentation, which may not become apparent until the blastocyst stage [25]. Hence, besides cleavage and blastocyst formation rates, the quality of developing IVC embryos after IVF is also important for evaluation of maturation events.

In the present study we have analyzed the presence of components of the Fas signaling pathway by examining their mRNA and protein expression. Furthermore, we have analyzed the function of the Fas signaling pathway by activating this system in cumulus cells during IVM of selected bovine COCs in the presence and absence of FSH. We also evaluated the effect of activating the Fas signaling pathway in cumulus cells on oocyte maturation and subsequent embryo development following IVF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of COCs

Bovine ovaries were collected at a local abattoir from cows of unknown reproductive status, kept at 30°C, and transported to the laboratory within 2 h after slaughter. COCs were aspirated from 3- to 8-mm antral follicles. Only those oocytes with homogeneous cytoplasm and a multilayered, compact cumulus investment [26] were used in the experiments.

In Vitro Maturation

COCs were rinsed with Hepes-buffered M199 (Gibco BRL, Paisley, U.K.) + 1% (v/v) penicillin-streptomycin (Gibco) to completely remove follicular fluid. In each of the three replicates, groups of 35 COCs were randomly allocated to each well of four-well culture plates (Nunc A/S, Roskilde, Denmark) containing 500 µl of the corresponding maturation medium of the different treatment groups: I, M199; II, M199 + 200 ng/ ml agonistic anti-Fas antibody (CH-11, Transduction Laboratories, Lexington, KY); III, M199 + 0.01 IU/ml recombinant hFSH (Organon, Oss, The Netherlands); IV, M199 + 200 ng/ml agonistic anti-Fas antibody + 0.01 IU/ml recombinant hFSH. IVM took place for 22 h at 39°C in humidified air containing 5% CO2.

Assessment of Oocyte Nuclear Maturation

Sample COCs were analyzed for nuclear maturation as previously described [27]. Briefly, oocytes were freed from their cumulus cells by vortexing and fixed in 2.5% (w/v) glutaraldehyde (Merck, Darmstadt, Germany) in PBS (Gibco). After fixation the oocytes were rinsed in PBS and stained with 0.285 µM 4,6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO) in PBS and mounted on slides. Oocytes were classified into four categories: degenerated (DEG), germinal vesicle (GV), metaphase I (MI), or metaphase II (MII) according to their nuclear morphology [28] as assessed by epifluorescence microscopy (BH2-RFCA; Olympus, Tokyo, Japan).

Assessment of Apoptosis in Cumulus Cells

Cell viability in COCs was assessed using a LIVE/DEAD kit (Viability/Cytotoxicity, Molecular Probes, Eugene, OR) according to the manufacturer's instructions, and nuclear morphology following incubation with Hoechst 33342 (Sigma). COCs were incubated for 10 min (39°C, 5% CO2 in humidified air) in 500 µl of PBS containing 2 µM ethidium homodimer-1 (EthD-1), 1 µM calcein-AM, and 10 µM Hoechst 33342; rinsed; mounted on slides; and immediately evaluated by epifluorescence microscopy using the following filters: 568 nm (EthD-1), 488 nm (calcein-AM), and 350 nm (Hoechst). A minimum of 200 cumulus cells per COC (3000 cells per treatment group) were randomly counted using a 1 cm2 grid inserted in the eyepiece of the microscope. Cells were evaluated for morphological characteristics of apoptotic cell death: chromatin condensation and nuclear fragmentation [29, 30]. Samples were also analyzed for imaging by multiphoton laser scanning microscopy combined with a confocal laser scanning microscope (CLSM; Leica TCS-SP, Heidelberg, Germany). Imaging was performed using a 488-nm Argon-ion laser and a 543-nm helium-neon laser to excite calcein-AM and EthD-1; Hoechst staining was imaged using a 100fs pulsed 780-nm excitation laser source (Spectra Physics, Mountain View, CA).

IVF, Embryo Culture, and Assessment of Blastocyst Quality

After maturation, oocytes in COCs were fertilized in vitro according to the procedure described by Parrish et al. [31], with minor modifications [32] using frozen-thawed semen from a bull of proven fertility. The presumptive zygotes were freed from cumulus cells 20 h after IVF by vortexing, and a maximum of 10 zygotes was placed in a 20-µl droplet of synthetic oviductal fluid (SOF) medium [33] supplemented with essential and nonessential amino acids (Sigma) and 0.1% (w/v) BSA (Sigma) under oil (Reproline Medical GmBH, Rheinbach, Germany) and cultured at 39°C in humidified air containing 5% CO2 and 7% O2. On Day 4 after IVF the number of cells per cleaved embryo was scored, and all cleaved embryos were transferred to fresh SOF droplets. The developmental stage of the embryos was assessed at Day 7 after IVF. Embryos that had not attained the blastocyst stage on Day 7 were excluded from further analysis.

All blastocysts obtained on Day 7 postfertilization were analyzed as described previously for assessment of the total number of cells per embryo (determined by nuclear staining with Hoechst 33342), as well as for the proportion of apoptotic cells (nuclei with fragmented DNA, positive for TUNEL and negative for EthD-1), and dead cells (positive for EthD-1) [34].

Extraction of Total RNA and Reverse Transcription-Polymerase Chain Reaction

Cumulus cells were separated from the oocytes immediately after collection using a narrow-bore Pasteur pipette. Denuded oocytes and cumulus cells were rinsed in PBS and stored at –80°C until RNA extraction. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) were performed separately on samples of 10 denuded oocytes and on their corresponding cumulus cells. Isolation of total RNA combined with on-column DNase digestion was performed using the RNeasy Mini Kit and the RNase-free DNase Set (Qiagen, Valencia, CA) according to the manufacturer's instructions. The RNA was eluted with 30 µl of RNase-free water. Complementary DNA synthesis and PCR were performed as previously described [27]. Minus RT blanks were prepared under the same conditions but without reverse transcriptase. Primers used for amplification are presented in Table 1. The thermal cycling profile was as follows: initial denaturation for 15 min at 94°C followed by 40 cycles of 15 sec at 94°C, 30 sec at 55°C, and 45 sec at 72°C. Final extension was for 10 min at 72°C. Ten microliters of the PCR product was separated by electrophoresis in 1% agarose gels containing 0.4 µg/ml ethidium bromide. A 100-base pair (bp) DNA ladder (Invitrogen, Breda, The Netherlands) was included as reference. The identity of the amplified products was verified by restriction enzyme digestion.


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  		TABLE 1. Primers used for PCR analysis

Indirect Immunofluorescence

Ten selected COCs and 10 denuded oocytes were fixed in 4% paraformaldehyde (Merck) in PBS overnight, rinsed in Tris-buffered saline (TBS: 50 mM Tris [ICN Biomedicals, Aurora, OH], 150 mM NaCl [Merck] pH 7.5), permeabilized in 0.1% Triton X-100 (Sigma) in TBS for 5 min, rinsed in TBS, and preincubated in 10% goat serum in TBS for 30 min at room temperature. COCs and oocytes were incubated overnight at 4°C with a polyclonal antibody against human Fas (sc-715; Santa Cruz Biotechnology, SanverTech, Heerhugowaard, The Netherlands) diluted 1: 100 in TBS–0.05% (w/v) acetylated BSA (Aurion, Wageningen, The Netherlands) (TBS-BSAc) or with normal rabbit serum diluted 1:1000 in TBS-BSAc (negative control). Samples were rinsed in TBS and incubated with a 1:300 goat-anti-rabbit immunoglobulin G (IgG) coupled to Alexa Fluor 488 (Molecular Probes) in TBS-BSAc for 1 h, rinsed in TBS, mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA), and analyzed using a 488-nm argon-ion laser CLSM (Leica).

SDS-PAGE and Western Blot Analysis

Samples consisted of oocytes, cumulus cells, and granulosa cells from freshly obtained ovaries (0 h), and of oocytes and cumulus cells isolated from COCs that had been cultured for 22 h in M199. An average of 800 oocytes and their corresponding cumulus cells were isolated per sample. In each of the replicates the cells were lysed in 150 µl of lysis buffer (20 mM Tris, 150 mM NaCl, 1% Triton X-100, 10% glycerol [Sigma], protease inhibitor cocktail [Roche, Mannheim, Germany], pH 7.4). Lysates were diluted 1:1 in sample buffer (62.5 mM Tris, 2% SDS, 10% glycerol, 1% ß-mercaptoethanol [Sigma], and 0.003% bromophenol blue [Sigma], pH 6.8), boiled for 10 min, followed by separation on 12% SDS-polyacrylamide gels, and transferred onto nitrocellulose membranes (Protan, Schleicher & Schuell GmBH, Dassel, Germany). The membranes were rinsed in TBS-Tween (TTBS: TBS, 0.1% Tween-20 [Merck]) and blocked with 5% nonfat dry milk in TTBS (blocking buffer) for 1 h, followed by overnight incubation at 4°C with a polyclonal antibody against human Fas (sc-715; Santa Cruz Biotechnology), polyclonal antibody against human FasL (sc-956; Santa Cruz Biotechnology), or a polyclonal antibody that recognizes both human procaspase-3 and active caspase-3 (Cell Signaling Technology, Westburg BV, Leusden, The Netherlands) diluted 1:500 in blocking buffer, or a polyclonal antibody against human actin (sc-1616; Santa Cruz Biotechnology) diluted 1:100 in blocking buffer. The membranes were rinsed with TTBS and incubated for 1 h with either horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Nordic Immunology, Tilburg, The Netherlands; Fas, FasL, caspase-3) diluted 1:10 000 in blocking buffer or with HRP-conjugated rabbit anti-goat IgG (Nordic Immunology; actin) diluted 1:500. Membranes were again rinsed in TTBS and TBS and, subsequently, the antibody-protein complexes were visualized using a supersignal chemiluminescent substrate (Pierce, Rockford, IL) and exposure to x-ray film (Fuji, Dusseldorf, Germany). As a control for antibody specificity, antibodies were also preincubated for 2 h with a 5x weight excess of the respective peptide used for immunization.

Statistical Analysis

Statistical analyses were conducted with SPSS software version 10.1 (SPSS, Chicago, IL), using an analysis of logistic regression [35] following a binomial distribution. Data from the evaluation of apoptosis in cumulus cells, oocyte nuclear maturation, cleavage, and blastocyst formation rates were analyzed using the following model: Ln ({pi}/1 – {pi}) = {alpha} + Fas + FSH + Fas*FSH, where {pi} represents the frequency of positive outcome, and {alpha} represents the intercept. Fas and FSH are independent categorical variables in this model. Data from blastocyst cell numbers were analyzed by one-way analysis of variance. Statistical differences were considered to be significant at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fas, FasL, and Caspase-3 Expression in COCs

To examine whether apoptosis could be induced via the Fas signaling pathway in bovine cumulus cells and oocytes, expression of FasL and Fas mRNA was examined by RT-PCR. FasL and Fas mRNA expression was detected in both cumulus cells and oocytes (Fig. 1). The presence of Fas and FasL protein was examined in samples from granulosa cells, cumulus cells, and oocytes. Immunoblot analysis of lysates performed with an antibody directed against human Fas yielded a band at the size of approximately 60 kDa (Fig. 2). Specificity of the observed band was demonstrated by preincubation of the antibody with its immunizing peptide, after which the band could no longer be detected (data not shown). Fas protein was found to be present in granulosa cells, cumulus cells, and oocytes. FasL protein was found in granulosa cells and cumulus cells but could not be detected in oocytes, despite mRNA expression (Fig. 2). Two bands were observed for FasL, most likely representing mature FasL and cleaved FasL [36]. Procaspase-3 protein was also found to be expressed in cumulus cells and in granulosa cells, but could not be detected in oocytes (Fig. 2). Procaspase-3 levels were higher in samples of freshly collected cumulus cells than in samples from cumulus cells cultured in vitro for 22 h, in which case only a faint band was revealed in some samples (data not shown). Protein loading was confirmed by immunoblotting of the same samples with an anti-ß-actin antibody (Fig. 2).



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  		FIG. 1. Expression of FasL and Fas mRNA in oocytes and cumulus cells as detected by RT-PCR. Samples are indicated at the top and a 100-bp DNA ladder is indicated for fragment size. Amplification of –RT blanks was performed as control



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  		FIG. 2. Western blot analysis of cell lysates from oocytes, cumulus cells, and granulosa cells at 0 h postcollection, and oocytes and cumulus cells from COCs after 22 h of IVM. Migration of molecular mass markers is indicated on the left

Fas protein expression was also detected in oocytes and cumulus cells by indirect immunofluorescence, confirming immunoblot findings. Fas expression was equally distributed between the peripheral and internal layers of the cumulus investment (Fig. 3A). In the oocyte, Fas expression was localized at the cellular membrane and its adjacent cytoplasmic region (Fig. 3B).



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  		FIG. 3. Confocal laser scanning images of a cumulus oocyte complex (A) and an oocyte (B) at 0 h postcollection following indirect immunofluorescence labeling for Fas (green). o, oocyte; cc, cumulus cells; zp, zona pellucida. Bar = 40 µm

Apoptosis in Cumulus Cells

To further elucidate the role of apoptosis in COCs, the Fas pathway was activated artificially by stimulation with the agonistic anti-Fas antibody CH-11 [37–40] at a concentration demonstrated (by dose-response experiments) to provide a maximum response. COCs were cultured for 22 h, after which the percentages of cells containing chromatin condensation were analyzed as a measure for apoptosis. Selected COCs showed a low apoptotic or nonapoptotic cumulus investment at the onset of IVM (Fig. 4A). COCs cultured in M199 maturation medium supplemented with either the agonistic anti-Fas antibody, FSH, or both, had higher rates of cumulus cells showing chromatin condensation indicative of apoptosis as compared to COCs cultured in M199 (Fig. 5). Poor quality COCs discarded from the experiments were also analyzed and showed positive staining for EthD-1, representing cellular death, affecting the whole cumulus investment homogeneously (Fig. 4B). After IVM of selected (i.e., good quality) COCs, the distribution of cumulus cells with signs of apoptosis in all treatment groups showed an irregular pattern with a higher incidence at the middle and peripheral layers of the complex, and fewer or no signs of apoptosis in corona radiata cells (Fig. 4, C and D). Signs of cumulus expansion were observed only in COCs cultured in the presence of FSH. The observed increase in apoptosis in cumulus cells from COCs cultured with the agonistic anti-Fas antibody suggests a complete Fas signaling pathway that induces apoptosis in cumulus cells. No protective effect of FSH toward apoptosis in cumulus cells was observed; on the contrary, in the presence of FSH during IVM the rate of apoptosis in these cells was significantly increased (Fig. 5).



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  		FIG. 4. Confocal laser scanning images of a good-quality COC (A) and an expanded poor-quality COC (B). Multiphoton laser scanning images of bovine COCs matured for 22 h in the presence (C) or absence (D) of FSH. Nuclei are stained with Hoechst (blue), nuclei from dead cells (arrowheads) are stained with EthD-1 (red), and viable cytoplasm is stained with calcein-AM (green). Cytoplasmic transzonal processes from the corona radiata cells can be observed (arrows) maintaining cell-to-cell contact with the oocyte. Bar = 40 µm (A, B), and 20 µm (C, D)



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  		FIG. 5. Average percentage of apoptotic cumulus cells after 22 h of IVM, of COCs cultured in different maturation media: I, M199; II, M199 + agonistic anti-Fas antibody; III, M199 + FSH; IV, M199 agonistic anti-Fas antibody + FSH. Values on bars with different labels a,b,c differ significantly (P ≤ 0.05)

Oocyte Nuclear Maturation

COCs and denuded oocytes that were subjected to IVM in the presence of the agonistic anti-Fas antibody with or without FSH were further analyzed for the assessment of nuclear maturation. In all treatment groups oocytes overcame meiotic arrest and reached the metaphase II (MII) stage of development after 22 h of incubation, matching routine IVM nuclear maturation rates [27, 41] (Table 2). IVM of COCs in the presence of FSH further enhanced the resumption of meiosis, diminishing the percentage of oocytes in the GV stage at 22 h of IVM as compared to oocytes matured in M199 or M199 supplemented with the agonistic anti-Fas antibody (Table 2). No significant differences were observed between the average percentages of degenerated oocytes or oocytes in MI or MII stages among COCs cultured in the presence or absence of the agonistic anti-Fas antibody, FSH, or both. Denuded oocytes that were subjected to IVM in the presence of the agonistic anti-Fas antibody with or without FSH showed no evident signs of apoptosis and exhibited no differences in nuclear maturation rates between treatment groups (Table 3). Combined, these observations suggest that the Fas signaling pathway in oocytes is incomplete and that induction of apoptosis in cumulus cells does not significantly affect nuclear maturation of the enclosed oocyte.


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  		TABLE 2. Stages of oocyte nuclear maturation in COCs from the different treatment groups after 22 h of IVM.*


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  		TABLE 3. Stages of oocyte nuclear maturation in denuded oocytes from the different treatment groups after 22 h of IVM.*

Blastocyst Yield and Blastocyst Quality

The effect of Fas-induced apoptosis in cumulus cells on the developmental capacity of the matured oocytes during IVF and IVC was also studied. COCs were in vitro matured in the presence of the agonistic anti-Fas antibody CH-11, with or without FSH, and subsequently followed identical IVF and IVC procedures. No significant differences were observed in cleavage rates between embryos developed from oocytes matured in vitro in the presence or absence of the agonistic anti-Fas antibody, FSH, or both (Fig. 6A). The presence or absence of the Fas-activating agent during IVM did not cause differences in blastocyst formation. Conversely, the blastocyst rate was significantly higher in the groups supplemented with FSH during IVM than in those cultured in M199 alone or M199 with the agonistic anti-Fas antibody (Fig. 6B). No significant differences were observed in the total number of cells per embryo (Fig. 7A), nor in the number of damaged cells (positive for TUNEL, EthD-1, or both) per embryo, among the treatment groups (Fig. 7B). Combined, these results indicate that induction of apoptosis in cumulus cells during IVM does not affect the developmental capacity of the enclosed oocyte.



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  		FIG. 6. Average percentages (referring to the total number of oocytes from each IVM treatment group, as indicated within the bars) ± SD, of (A) embryos cleaved at Day 4 (cleavage rate), and (B) blastocysts obtained at Day 7 (blastocyst rate) in all treatment groups. I, IVM in M199; II, IVM in M199 + agonistic anti-Fas antibody; III, IVM in M199 + FSH; IV, IVM in M199 + agonistic anti-Fas antibody + FSH. a,bValues on bars differ significantly (P ≤ 0.05)



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  		FIG. 7. A) Total number of cells per embryo (nuclei stained with Hoechst) (mean ± SD) and (B) damaged cells (positive for EthD-1, TUNEL, or both) per blastocyst obtained at Day 7 from all treatment groups. I, IVM in M199; II, IVM in M199 + agonistic anti-Fas antibody; III, IVM in M199 + FSH; IV, IVM in M199 agonistic anti-Fas antibody + FSH. No significant differences were observed between treatment groups. The number of blastocysts is indicated within the bars


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Folliculogenesis is a complex process, the primary function of which is the preparation of an oocyte for ovulation and subsequent fertilization. Simultaneously, however, during each wave of follicular development in each estrous cycle, many follicles start to develop, of which most will eventually undergo atresia. Although follicular cell death is predominantly mediated via apoptosis [42], the precise mechanisms by which these processes are regulated are largely unknown. Here we have examined the involvement of the Fas signaling pathway in apoptotic events that take place in bovine COCs originated from antral follicles and have demonstrated that this pathway is functional in the cumulus cells but not in the oocytes. Fas-induced apoptosis in cumulus cells did not, however, lead to differences in oocyte maturation, nor subsequent embryo development following fertilization.

In both oocytes and cumulus cells, expression of Fas mRNA and protein was detected by RT-PCR and indirect immunofluorescence. Western blot analysis of cell lysates from cumulus cells and oocytes using an antibody against human Fas revealed a band in all samples at approximately 60 kDa, and not at the expected 45 kDa. Specificity of this band was determined by preincubation of the antibody with an excess of its immunizing peptide that prevented antibody binding to the 60 kDa protein. The size of bovine Fas mRNA is similar to that of the human [43], and therefore, the protein is expected to be of similar size. Fas is believed to aggregate in trimeric proteins at the cell surface, but the observed 60 kDa product is not likely a result of aggregation, because this would lead to protein bands with a molecular mass of approximately 200 kDa [44]. However, because Fas contains multiple glycosylation sites [38], the observed 60 kDa band may represent a glycosylated form of Fas. This heterogeneity of the Fas protein has also been observed in human cell lines, providing different sized proteins in different cell lines [44]. The observed expression of Fas in bovine oocytes is in coincidence with observations in humans [45], but in contrast to those in mice [46], in which no expression of Fas protein was found in oocytes.

Imunoblot analysis with an antibody directed against FasL revealed two bands of approximately 40 and 22 kDA in lysates from granulosa and cumulus cells. Specificity of these bands was also determined by preincubating the antibody with its immunizing peptide. These bands are similar to the reported sizes of wild-type (37 to 43 kDa) [36, 47, 48] and soluble (18 to 29 kDa) FasL [15, 49], and probably represent the mature and cleaved forms of FasL [36]. The variability in the expression of FasL observed among species is of interest. FasL mRNA or protein expression could not be detected in mouse oocytes [50]. In the present study, FasL mRNA was expressed in bovine oocytes, but this transcript was not translated or was translated at such a low level that it could not be detected by immunoblotting, similar to what has been observed in human oocytes [15]. However, both FasL mRNA and protein expression have been observed in rat oocytes [51]. It is unlikely that the observed FasL mRNA expression in oocytes resulted from contamination with cumulus cells, because for mRNA isolation only, oocytes were used that were individually and completely denuded. Approximately 8 h after GV breakdown, transcription of the oocyte genome is stopped until after fertilization, and major activation of embryonic transcription starts then at the 8- to 16-cell stage [52]. It is therefore possible that FasL mRNA is transcribed in the oocyte, to be translated later when necessary.

In the present study Fas, FasL, and procaspase-3 were demonstrated to be expressed in cumulus cells, suggesting a functional Fas signaling pathway in these cells, as has also been demonstrated in granulosa and theca cells [3, 15]. Procaspase-3 protein levels were considerably reduced in cumulus cells after IVM for 22 h. Nevertheless, artificial stimulation of the Fas signaling pathway in cumulus cells using an agonistic anti-Fas antibody resulted in a significant increase in the number of apoptotic cumulus cells. This increase of apoptosis in cumulus cells did not affect the developmental potential of the enclosed oocytes, as evaluated in terms of nuclear maturation, cleavage, or blastocyst formation rates, nor blastocyst quality following fertilization.

In the follicle, the COC is the last compartment that is affected by apoptosis [18, 53]. A strong correlation has been observed between morphological grading of COCs and apoptosis in cumulus cells. Poor morphology (grade 3) COCs have more apoptotic cumulus cells than COCs in which the oocytes have an homogeneous cytoplasm and are surrounded by a complete and compact cumulus investment (grade 1 COCs) [17, 54]. It is interesting that oocytes from slightly atretic COCs showing signs of cumulus expansion (grade 2) have a better embryonic developmental capacity after IVF than those considered to be of the highest quality (grade 1) [18, 26]. Concordantly, we observed an increase in the percentage of apoptotic cumulus cells when COCs were exposed to FSH, while simultaneously, oocytes from these COCs exhibited a higher developmental potential in terms of blastocyst formation rate in accordance with previous reports [23, 24]. FSH induces cumulus expansion, leading to a progressive uncoupling of the middle and peripheral cumulus cells, and interestingly, it is in these regions of the cumulus investment that we observed the highest incidence of apoptosis, as has also been reported in vivo in rat COCs [55]. The loss of cell-to-cell contact coinciding with cumulus expansion could trigger apoptosis in these cells, similar to what has been observed in mural granulosa cells [56]. The enhanced developmental capacity of oocytes from COCs exposed to FSH on the other hand must be attributed to an indirect effect of FSH through the cumulus cells, because oocytes lack receptors for this hormone [24].

Of the cumulus cells, the corona radiata cells exhibited the lowest incidence of apoptosis. These cells also remained tightly attached to the oocyte even after stimulation with FSH, and could thus support oocyte nuclear and cytoplasmic maturation [16]. Therefore a low cell viability in the corona radiata cell as has been observed in low-quality COCs could negatively affect the developmental capacity of the oocyte. A limited level of apoptosis in the peripheral cumulus on the other hand could facilitate sperm penetration.

In oocytes, Fas mRNA and protein expression were detected, whereas FasL and caspase-3 protein expression could not be detected, suggesting that the Fas signaling pathway may not be active in bovine oocytes, similar to what has been suggested in human [15, 57] and mouse oocytes [13, 14]. Indeed, stimulation of Fas with an agonistic antibody in denuded bovine oocytes during IVM did not lead to any differences in nuclear maturation rates compared to control denuded oocytes, nor was apoptosis observed in these oocytes. On the other hand, exposure of oocytes to staurosporine, a general inducer of apoptosis [58], did lead to evident signs of apoptosis such as nuclear condensation and fragmentation (data not shown), demonstrating that bovine oocytes can undergo apoptosis in vitro, although through a mechanism that most likely does not involve activation of Fas. The activating processes of apoptosis in oocytes need to be further investigated.

In conclusion, apoptosis in bovine cumulus cells during IVM can be induced via activation of the Fas signaling pathway, a pathway that is not functional in oocytes. Moderate apoptosis in cumulus cells during IVM as induced by Fas signaling or FSH does not severely affect the corona radiata cells and has no evident unfavorable effect on the developmental potential of the oocyte.


   FOOTNOTES
 
1 This work was supported in part by funds from Holland Genetics. Back

2 Correspondence: Bernard A.J. Roelen, Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 7, 3584 CL Utrecht. FAX: 31 030 253 4811; b.a.j.roelen{at}vet.uu.nl Back

Received: 17 February 2003.

First decision: 9 March 2004.

Accepted: 23 April 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Garcia A, van der Weijden GC, Colenbrander B, Bevers MM. Monitoring follicular development in cattle by real-time ultrasonography: a review. Vet Rec 1999 145:334-340[Abstract/Free Full Text]
  2. Hendriksen PJ, Gadella BM, Vos PL, Mullaart E, Kruip TA, Dieleman SJ. Follicular dynamics around the recruitment of the first follicular wave in the cow. Biol Reprod 2003 69:2036-2044[Abstract/Free Full Text]
  3. Foghi A, Ravandi A, Teerds KJ, Van Der Donk H, Kuksis A, Dorrington J. Fas-induced apoptosis in rat thecal/interstitial cells signals through sphingomyelin-ceramide pathway. Endocrinology 1998 139:2041-2047[Abstract/Free Full Text]
  4. Yang MY, Rajamahendran R. Morphological and biochemical identification of apoptosis in small, medium, and large bovine follicles and the effects of follicle-stimulating hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured bovine granulosa cells. Biol Reprod 2000 62:1209-1217[Abstract/Free Full Text]
  5. Tajima K, Orisaka M, Hosokawa K, Amsterdam A, Kotsuji F. Effects of ovarian theca cells on apoptosis and proliferation of granulosa cells: changes during bovine follicular maturation. Biol Reprod 2002 66:1635-1639[Abstract/Free Full Text]
  6. Vickers SL, Cowan RG, Harman RM, Porter DA, Quirk SM. Expression and activity of the Fas antigen in bovine ovarian follicle cells. Biol Reprod 2000 62:54-61[Abstract/Free Full Text]
  7. Porter DA, Harman RM, Cowan RG, Quirk SM. Relationship of Fas ligand expression and atresia during bovine follicle development. Reproduction 2001 121:561-566[Abstract]
  8. Hu CL, Cowan RG, Harman RM, Porter DA, Quirk SM. Apoptosis of bovine granulosa cells after serum withdrawal is mediated by Fas antigen (CD95) and Fas ligand. Biol Reprod 2001 64:518-526[Abstract/Free Full Text]
  9. Thorburn A. Death receptor-induced cell killing. Cell Signal 2004 16:139-144[CrossRef][Medline]
  10. Wajant H. The Fas signaling pathway: more than a paradigm. Science 2002 296:1635-1636[Abstract/Free Full Text]
  11. Matikainen T, Perez GI, Zheng TS, Kluzak TR, Rueda BR, Flavell RA, Tilly JL. Caspase-3 gene knockout defines cell lineage specificity for programmed cell death signaling in the ovary. Endocrinology 2001 142:2468-2480[Abstract/Free Full Text]
  12. Vaux DL, Korsmeyer SJ. Cell death in development. Cell 1999 96:245-254[CrossRef][Medline]
  13. Sato E. Morphological dynamics of cumulus-oocyte complex during oocyte maturation. Ital J Anat Embryol 1998 103:103-118[Medline]
  14. Dharma SJ, Kelkar RL, Nandedkar TD. Fas and Fas ligand protein and mRNA in normal and atretic mouse ovarian follicles. Reproduction 2003 126:783-789[Abstract]
  15. Jose de los Santos M, Anderson DJ, Racowsky C, Hill JA. Presence of Fas-Fas ligand system and bcl-2 gene products in cells and fluids from gonadotropin-stimulated human ovaries. Biol Reprod 2000 63:1811-1816[Abstract/Free Full Text]
  16. Tanghe S, Van Soom A, Mehrzad J, Maes D, Duchateau L, de Kruif A. Cumulus contributions during bovine fertilization in vitro. Theriogenology 2003 60:135-149[CrossRef][Medline]
  17. Zeuner A, Muller K, Reguszynski K, Jewgenow K. Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation. Theriogenology 2003 59:1421-1433[CrossRef][Medline]
  18. de Wit AA, Wurth YA, Kruip TA. Effect of ovarian phase and follicle quality on morphology and developmental capacity of the bovine cumulus-oocyte complex. J Anim Sci 2000 78:1277-1283[Abstract/Free Full Text]
  19. Hendriksen PJ, Vos PL, Steenweg WN, Bevers MM, Dieleman SJ. Bovine follicular development and its effect on the in vitro competence of oocytes. Theriogenology 2000 53:11-20[CrossRef][Medline]
  20. Host E, Gabrielsen A, Lindenberg S, Smidt Jensen S. Apoptosis in human cumulus cells in relation to zona pellucida thickness variation, maturation stage, and cleavage of the corresponding oocyte after intracytoplasmic sperm injection. Fertil Steril 2002 77:511-515[CrossRef][Medline]
  21. Lee KS, Joo BS, Na YJ, Yoon MS, Choi OH, Kim WW. Cumulus cells apoptosis as an indicator to predict the quality of oocytes and the outcome of IVF-ET. J Assist Reprod Genet 2001 18:490-498[CrossRef][Medline]
  22. Ikeda S, Imai H, Yamada M. Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes. Reproduction 2003 125:369-376[Abstract]
  23. Izadyar F, Zeinstra E, Bevers MM. Follicle-stimulating hormone and growth hormone act differently on nuclear maturation while both enhance developmental competence of in vitro matured bovine oocytes. Mol Reprod Dev 1998 51:339-345[CrossRef][Medline]
  24. van Tol HT, van Eijk MJ, Mummery CL, van den Hurk R, Bevers MM. Influence of FSH and hCG on the resumption of meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa. Mol Reprod Dev 1996 45:218-224[CrossRef][Medline]
  25. Watson AJ, De Sousa P, Caveney A, Barcroft LC, Natale D, Urquhart J, Westhusin ME. Impact of bovine oocyte maturation media on oocyte transcript levels, blastocyst development, cell number, and apoptosis. Biol Reprod 2000 62:355-364[Abstract/Free Full Text]
  26. Gordon I. . Laboratory Production of Cattle Embryos, 2nd Ed. Wallingford, U.K.: CABI Publishing; 2003:79–111
  27. Beker-Van Woudenberg AR, van Tol HT, Roelen BA, Colenbrander B, Bevers MM. Estradiol and its membrane-impermeable conjugate (estradiol-bovine serum albumin) during in vitro maturation of bovine oocytes: effects on nuclear and cytoplasmic maturation, cytoskeleton, and embryo quality. Biol Reprod 2004 70:1465-1474[Abstract/Free Full Text]
  28. Mori C, Hashimoto H, Hoshino K. Fluorescence microscopy of nuclear DNA in oocytes and zygotes during in vitro fertilization and development of early embryos in mice. Biol Reprod 1988 39:737-742[Abstract]
  29. Sakahira H, Enari M, Ohsawa Y, Uchiyama Y, Nagata S. Apoptotic nuclear morphological change without DNA fragmentation. Curr Biol 1999 9:543-546[CrossRef][Medline]
  30. Renvoize C, Biola A, Pallardy M, Breard J. Apoptosis: identification of dying cells. Cell Biol Toxicol 1998 14:111-120[CrossRef][Medline]
  31. Parrish JJ, Susko Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988 38:1171-1180[Abstract]
  32. Izadyar F, Zeinstra E, Colenbrander B, Vanderstichele HM, Bevers MM. In vitro maturation of bovine oocytes in the presence of bovine activin A does not affect the number of embryos. Anim Reprod Sci 1996 45:37-45[CrossRef][Medline]
  33. van Wagtendonk de Leeuw AM, Mullaart E, de Roos AP, Merton JS, den Daas JH, Kemp B, de Ruigh L. Effects of different reproduction techniques: AI MOET or IVP, on health and welfare of bovine offspring. Theriogenology 2000 53:575-597[CrossRef][Medline]
  34. Pomar FJ, Ducro-Steverink DW, Hazeleger W, Teerds KJ, Colenbrander B, Bevers MM. Development, DNA fragmentation and cell death in porcine embryos after 24 h storage under different conditions. Theriogenology 2004 61:147-158[CrossRef][Medline]
  35. Mc Cullagh PNJ. Binary Data. Generalized Linear Models. London: Chapman & Hall; 1989
  36. Schneider P, Bodmer JL, Holler N, Mattmann C, Scuderi P, Terskikh A, Peitsch MC, Tschopp J. Characterization of Fas (Apo-1, CD95)-Fas ligand interaction. J Biol Chem 1997 272:18827-18833[Abstract/Free Full Text]
  37. Quirk SM, Cowan RG, Joshi SG, Henrikson KP. Fas antigen-mediated apoptosis in human granulosa/luteal cells. Biol Reprod 1995 52:279-287[Abstract]
  38. Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima S, Sameshima M, Hase A, Seto Y, Nagata S. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 1991 66:233-243[CrossRef][Medline]
  39. Watson RW, Rotstein OD, Parodo J, Jimenez M, Soric I, Bitar R, Marshall JC. Impaired apoptotic death signaling in inflammatory lung neutrophils is associated with decreased expression of interleukin-1 beta converting enzyme family proteases (caspases). Surgery 1997 122:163-171 discussion 171–172 [CrossRef][Medline]
  40. Yonehara S. Effects of anti-Fas antibodies on lymphocytes and other organs: preparation of original and new monoclonal antibodies and amelioration of systemic autoimmune disease. Int Rev Immunol 1999 18:329-345[Medline]
  41. Hashimoto S, Saeki K, Nagao Y, Minami N, Yamada M, Utsumi K. Effects of cumulus cell density during in vitro maturation of the developmental competence of bovine oocytes. Theriogenology 1998 49:1451-1463[CrossRef][Medline]
  42. Tilly JL. Apoptosis and ovarian function. Rev Reprod 1996 1:162-172[Abstract]
  43. Yoo J, Stone RT, Beattie CW. Cloning and characterization of the bovine Fas. DNA Cell Biol 1996 15:227-234[Medline]
  44. Kamitani T, Nguyen HP, Yeh ET. Activation-induced aggregation and processing of the human Fas antigen. Detection with cytoplasmic domain-specific antibodies. J Biol Chem 1997 272:22307-22314[Abstract/Free Full Text]
  45. Kondo H, Maruo T, Peng X, Mochizuki M. Immunological evidence for the expression of the Fas antigen in the infant and adult human ovary during follicular regression and atresia. J Clin Endocrinol Metab 1996 81:2702-2710[Abstract]
  46. Sakamaki K, Yoshida H, Nishimura Y, Nishikawa S, Manabe N, Yonehara S. Involvement of Fas antigen in ovarian follicular atresia and luteolysis. Mol Reprod Dev 1997 47:11-18[CrossRef][Medline]
  47. Orlinick JR, Elkon KB, Chao MV. Separate domains of the human fas ligand dictate self-association and receptor binding. J Biol Chem 1997 272:32221-32229[Abstract/Free Full Text]
  48. Peduto Eberl L, Guillou L, Saraga E, Schroter M, French LE, Tschopp J, Juillerat-Jeanneret L. Fas and Fas ligand expression in tumor cells and in vascular smooth-muscle cells of colonic and renal carcinomas. Int J Cancer 1999 81:772-778[CrossRef][Medline]
  49. Stokes TA, Rymaszewski M, Arscott PL, Wang SH, Bretz JD, Bartron J, Baker JR Jr, Fiedler P, Schaetzlein CE, Eibel H, Papoff G, Stassi G, De Maria R, Giordano C, Galluzzo A, Bagnasco M, Ruberti G, Testi R. Constitutive expression of FasL in thyrocytes. Science 1998 279:2015a
  50. Guo MW, Xu JP, Mori E, Sato E, Saito S, Mori T. Expression of Fas ligand in murine ovary. Am J Reprod Immunol 1997 37:391-398
  51. Hakuno N, Koji T, Yano T, Kobayashi N, Tsutsumi O, Taketani Y, Nakane PK. Fas/APO-1/CD95 system as a mediator of granulosa cell apoptosis in ovarian follicle atresia. Endocrinology 1996 137:1938-1948[Abstract]
  52. Viuff D, Avery B, Greve T, King WA, Hyttel P. Transcriptional activity in in vitro produced bovine two- and four-cell embryos. Mol Reprod Dev 1996 43:171-179[CrossRef][Medline]
  53. Kruip TA, Dieleman SJ. Macroscopic classification of bovine follicles and its validation by micromorphological and steroid biochemical procedures. Reprod Nutr Dev 1982 22:465-473
  54. Bilodeau Goeseels S, Panich P. Effects of oocyte quality on development and transcriptional activity in early bovine embryos. Anim Reprod Sci 2002 71:143-155[CrossRef][Medline]
  55. Szoltys M, Tabarowski Z, Pawlik A. Apoptosis of postovulatory cumulus granulosa cells of the rat. Anat Embryol (Berl) 2000 202:523-529[CrossRef][Medline]
  56. Peluso JJ, Pappalardo A, Trolice MP. N-cadherin-mediated cell contact inhibits granulosa cell apoptosis in a progesterone-independent manner. Endocrinology 1996 137:1196-1203[Abstract]
  57. Payne SG, Smith SC, Davidge ST, Baker PN, Guilbert LJ. Death receptor Fas/Apo-1/CD95 expressed by human placental cytotrophoblasts does not mediate apoptosis. Biol Reprod 1999 60:1144-1150[Abstract/Free Full Text]
  58. Zhang G, Yan G, Gurtu V, Spencer C, Kain SR. Caspase inhibition prevents staurosporine-induced apoptosis in CHO-K1 cells. Apoptosis 1998 3:27-33[CrossRef][Medline]




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