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Regular Article |
a Agrifood Research Finland, Animal Production Research, FIN-31600 Jokioinen, Finland
b Research Institute of Animal Production, SK-949 92 Nitra, Slovakia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, embryo, fertilization, growth factors, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In normally developing human morulae and blastocysts, some cells spontaneously undergo programmed cell death, or apoptosis, which might be involved in the elimination of abnormal cells [10]. Moreover, apoptosis may play a role in eliminating abnormal or defective embryos during the first cell cycle in mice [11] and during the second cell cycle in humans [12]. At the cellular level, apoptosis is characterized by cytoplasmic and nuclear fragmentation, chromatin condensation, DNA fragmentation, and phagocytosis [10, 13]. Nuclear changes typical for early stage apoptosis include DNA fragmentation, which can be detected using TUNEL. During in vitro embryo culture, apoptosis is probably caused by suboptimal conditions [14] and may therefore also be an indicator of embryo quality [15]. Apoptosis has been observed in bovine embryos after the 8-cell stage [15], when the bovine embryonic genome is activated [16]. The majority of apoptotic nuclei in bovine blastocysts were located in the inner cell mass (ICM) [15], whereas in humans [10] and mice [11] apoptotic cells were distributed randomly in the embryo.
Another parameter used to assess the quality of embryos is the presence of proliferating cell nuclear antigen (PCNA), which is a specific marker of the cell cycle S-phase [17] and an essential component of the DNA replication and repair machinery [18]. Immunocytochemical detection of PCNA can be used for the evaluation of developmental potential of fresh [19] and frozen-thawed [20] bovine IVP embryos.
Supplementation of culture medium with IGF-I has a beneficial effect on preimplantation embryo development by decreasing apoptosis and increasing cell proliferation in mouse [21], rabbit [4], and human [6, 22] blastocysts. The antiapoptotic effect of IGF-I is mediated via an IGF-I receptor pathway, because reduction in the number of IGF-I receptors causes extensive apoptosis whereas IGF-I receptor overexpression protects cells from apoptosis in vivo [23]. However, there have been no studies describing the effects of IGF-I supplementation on the apoptotic and proliferation index measured as TUNEL- and PCNA-positive cells in bovine IVP embryos.
The aim of the present study was to determine whether IGF-I acts as an antiapoptotic or a mitogenic factor during preimplantation development of bovine IVP embryos. We examined 1) the frequency of apoptosis using TUNEL assay, 2) the ratio of PCNA-positive nuclei to the total cell number, and 3) the relationship between the PCNA index and TUNEL index in Day 6.5 blastocysts obtained in the presence or absence of IGF-I in maturation and/or culture medium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Vitro Embryo Production
Ovaries from various cattle breeds were collected at a local slaughterhouse and transported to the laboratory within 8 h at room temperature. Follicular fluid containing the oocytes was aspirated by needle and syringe. All cumulus-oocyte complexes (COC), except those that were overmatured, were used for IVP [20]. COCs were washed in in vitro maturation (IVM) medium, TCM-199 with Glutamax (Gibco, Grand Island, NY), supplemented with FSH (2 µg/ml, Ovagen TM; Immuno-Chemical Products Ltd., Auckland, New Zealand), LH (10 µg/ml), estradiol (1 µg/ml), 25 mM sodium-pyruvic acid, 100 IU/ml penicillin G, 100 µg/ml streptomycin, and 5% fetal calf serum (FCS, Gibco BRL, Auckland, New Zealand). COCs were matured in groups of 50/500 µl IVM medium under mineral oil for 24 h at 39°C under 5% CO2 in humidified air.
Frozen-thawed and washed sperm from a single Ayrshire bull of proven in vitro fertility were used for fertilization. Spermatozoa (1.5 x 106 sperm/ml) and COCs (100–125 COCs/500 µl) were incubated in modified fert-TALP medium with 10 µg/ml heparin [24] for 20 h at 39°C under 5% CO2 in humidified air.
All presumptive zygotes were vortexed for 90 sec in Hepes-TALP to remove the cumulus cells, washed once in Hepes-TALP and twice in in vitro culture medium (IVC medium: synthetic oviductal fluid [SOF] supplemented with Eagle basal medium amino acids, modified Eagle medium amino acids, sodium citrate, and myo-inositol [SOFaaci] [25] and 5% FCS), and placed in groups of 50 zygotes in a 35-µl drop of culture medium under mineral oil in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 at 39°C. Human recombinant IGF-I was used in the experiments. In our preliminary study, both 50 and 500 ng/ml of IGF-I in the maturation medium significantly increased the blastocyst yield and the ratio of proliferating cells [26]. Therefore, in the present study, the middle concentration of 100 ng/ml of IGF-I was chosen. IGF-I was added to the maturation medium (100/0), the culture medium (0/100), or both media (100/100). Standard protocol with no IGF-I addition (0/0) was used as a control. The presumptive zygotes were cultured in the same drop without changing the medium for about 136 h (until Day 6.5). The cleavage percentage and blastocyst rates were calculated from the total number of oocytes. Embryos with a defined blastocoele were selected for immunocytochemical staining, apoptosis detection, and counterstaining.
PCNA Immunocytochemistry and TUNEL Assay
Embryos were washed (3 times for 5 min) in PBS supplemented with polyvinylpyrrolidone (PBS-PVP, 4 mg/ml) and then fixed for 5 min in neutral buffered formalin (3.7%) and for 10 min in 70% ethanol. For membrane permeabilization, embryos were incubated in 0.5% Triton X-100 in PBS for 15 min. Nonspecific binding of antibodies was suppressed by incubation in blocking solution (1% BSA in PBS). Staining for PCNA was combined with the detection of apoptosis (TUNEL apoptosis detection kit; Upstate Biotechnology, Lake Placid, NY).
Fixed embryos were incubated in TUNEL cocktail for 1 h. The reaction was stopped, and the embryos were washed and transferred into the drop of monoclonal anti-PCNA antibody (clone PC-10; DAKO, Glostrup, Denmark) at a dilution of 1:50 in blocking solution for 1 h. Apoptotic and proliferating cells were detected by incubating the embryos for 30 min in a mixture of avidin-fluorescein isothiocyanate (FITC) (1:10) and tetramethyl rhodamine isothiocyanate (TRITC)-labeled mouse immunoglobulin (1:50) (DAKO), respectively, in the blocking solution. Between incubations, embryos were washed 3 times for 5 min in PBS-PVP. After the last wash, embryos were put onto a coverslip and covered immediately with 6 µl of warm mounting medium (1% agar with 100 mg of 1,4-diazobicyclo-(2.2.2)octane (Dabco 1:1). The coverslip was mounted onto a slide with small drops of nail polish. After hardening of the mounting medium and nail polish, glycerol diluted with PBS (2:1) was applied carefully under the coverslip. The attachment of the coverslip was secured with nail polish. The slides were stored at -20°C until scanning, which was usually performed within 3 wk.
As a positive control for TUNEL, a group of fixed embryos was incubated in the presence of bovine DNase I (1 µl/ml in PBS) for 1 h at 37°C before the TUNEL reaction. Thereafter the protocol was continued as above. The specificity of TUNEL binding and PCNA binding was verified by omitting the TUNEL reagent or the PCNA antibody, respectively, from the protocol.
Laser Confocal Microscopy and Counting of TUNEL-, PCNA-, and Propidium Iodide-Positive Nuclei
Whole-mount embryos were studied with a Leica TCS NT laser confocal microscope using simultaneous detection for TUNEL (FITC channel, absorbance at 495 nm, emission at 525 nm) , and PCNA (TRITC channel, absorbance at 536 nm, emission at 623 nm); later, propidium iodide (PI) was detected using the TRITC channel. The 40 x 1.25 objective was used.
After confocal scanning of the embryos, the nail polish was removed from the edges of the coverslips, and the coverslips were carefully detached from the slides. The embryos were recovered from the mounting medium, washed 3 times in PBS-PVP solution, and counterstained with PI (200 µg/ml) in blocking solution for 30 min for determination of the total number of blastocysts. Embryos were mounted under the coverslips as described above and scanned repeatedly to detect red PI fluorescence.
The numbers of TUNEL-, PCNA-, and PI-positive nuclei were determined from optical section images, each representing a 0.8-µm optical section of the embryo, to obtain the data on apoptotic, proliferating, and total nuclei. The total number of nuclei per embryo could not be counted from the section because many nuclei were obviously present in several sections. Therefore, the numbers of TUNEL- and PI-positive nuclei from overlay stereoimages were also included. Location of apoptotic cells in different embryo compartments was defined from sections and overlay stereoimages. Because the confocal method was not supported by the differential staining of blastocyst cell lineages, the cells were classified according to the position, shape, and size of nuclei as belonging to the ICM area or the trophectoderm (TE) area.
Statistical Analysis
Differences between treatment groups were tested using a Kruskal-Wallis ANOVA. When strong evidence of a difference among treatments was found, more specific comparisons between groups were performed using a SAS macro [27]. The TUNEL:PI ratio, the total number of cells per blastocyst, and the number of TUNEL-positive cells per embryo were almost normally distributed. Nevertheless, the arcsine square root and square root transformations were used on the data for the ratio and the TUNEL-positive cells, respectively. These data were analyzed using a one-way ANOVA. A chi-square test for the contingency table was used for the data in Tables 1 and 4. Pearson correlation coefficients were used to identify associations between 2 variables. All statistical analyses were performed using SAS software (SAS Institute Inc., Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Altogether, 832 oocytes originating from 4 slaughterhouse batches of ovaries were used in this study. The addition of IGF-I to the IVM medium, the IVC medium, or both media did not affect significantly the cleavage rate (Table 1). The significant (P = 0.03) difference in the percentage of Day 6.5 morula/blastocyst stage embryos was found between the 0/0 and 0/100 groups, when the test without multiple-comparison adjustment was used for the estimation. There was no significant difference in the distribution of blastocysts according to developmental stage between any of the groups (data not shown).
Effect of IGF-I on Apoptosis and Total Cell Number in Day 6.5 Blastocysts
DNA fragmentation in blastocyst nuclei was revealed as a green fluorescence signal after specific binding with TUNEL reagent and FITC conjugate (Fig. 1, A and D). The embryos in which the TUNEL reagent was omitted showed no fluorescence signals (Fig. 1H), but the embryos preincubated with bovine DNase demonstrated TUNEL-positive reaction in all nuclei (Fig. 1G).
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The presence of IGF-I in the IVM medium, the IVC medium, or both media did not significantly reduce the frequency of blastocysts with apoptotic cells. However, when IGF-I was added to the IVC medium, the average number of apoptotic cells per embryo was significantly decreased (Table 2).
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Using the standard IVP procedure (0/0), about 5% of cells of the blastocysts were TUNEL positive. The addition of IGF-I into IVC medium (0/100 and 100/100) but not into IVM medium (100/0) reduced the TUNEL index to approximately 2.5% and significantly increased the total cell number of blastocysts (Table 3). A positive correlation between the number of TUNEL-positive cells and the total number of cells (r = 0.493) was found in the control group (0/0). After addition of IGF-I to IVM (r = 0.362) or to IVC (r = 0.384) media, the correlation coefficient decreased slightly, and there was only a slight correlation between these parameters after IGF-I addition to both IVM and IVC media (r = 0.262). We did not observe any significant differences between TUNEL indices obtained by counting either from optical sections or from overlay images in any group (data not shown). Moreover, we observed a high linear regression (r = 0.827, P = 0.0001) between the number of PI-positive nuclei counted from sections and the total number of cells counted from overlay stereoimages.
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Distribution of the TUNEL-positive nuclei in the embryo compartments was different for the control group (0/0) versus the groups with IGF-I in the IVC medium (0/100 and 100/100) but not versus the group with IGF-I in the IVM medium (100/0) (Table 4). In IVC medium (groups 0/100 and 100/100), IGF-I significantly decreased the percentage of embryos with apoptotic cells distributed within both the ICM and TE areas, whereas the proportion of embryos with apoptotic cells located exclusively in the ICM area was significantly increased when compared with either group 0/0 or group 100/0.
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Effect of IGF-I on PCNA-Positive Nuclei in Day 6.5 Blastocysts
The healthy embryos with undamaged membranes and without lysed (dark) cells, which were subjected to PCNA immunocytochemistry, showed clear red fluorescence in almost all nuclei (Fig. 1, B and C). Fluorescence was not seen in negative controls, where PCNA antibody was omitted (Fig. 1I). In the control group 0/0, almost 80% of nuclei were positive for PCNA. The addition of IGF-I to IVM medium, to IVC medium, or to both media resulted in a slight but not significant increase in the ratio of PCNA-positive nuclei (Fig. 2).
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Colocalization of TUNEL- and PCNA-Positive Reactions
Colocalization of TUNEL-positive and PCNA-positive fluorescence (Fig. 1, A–C) was observed in 60%–66% of all blastocysts. Between treatments, there was no significant difference in the percentage of TUNEL-positive cells that were also PCNA positive (Table 5). A positive, but not strong, correlation was observed between the TUNEL and PCNA reactions in the control group (0/0) and in the group where IGF-I was added to the IVM medium (100/0). However, when IGF-I was added to the IVC medium, the TUNEL/PCNA correlation was higher (Table 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Peptide growth factors play a crucial regulatory role during oocyte maturation and preimplantation development [28]. In particular, IGF-I is a potent stimulator of oocyte maturation and subsequent embryo development [2–4, 6] and cell proliferation [4, 6, 21] and is a suppressor of apoptosis [4, 22]. It is still unclear whether IGF-I is secreted in abundant amounts into the milieu by preimplantation embryos. The presence of IGF-I ligand mRNA at all embryo stages from the zygote to the blastocyst [9, 29, 30] was supported by colloidal-gold immunocytochemical detection of IGF-I peptide in bovine blastocysts [30], confirming the IGF-I synthetic ability; however, the release of embryonic IGF-I was below detection levels [31, 32]. In vivo, the maternal IGF-I present in ovarian follicular fluid [33] and in oviductal [34] and uterine [35] secretions provides an IGF-I-rich environment for the oocyte and the subsequent embryo [32].
We determined the effect of IGF-I in IVM medium, IVC medium, or both media on the development and functional parameters such as the PCNA and TUNEL index. Of all Day 6.5 blastocysts obtained in the present study, about 50% were blastocysts, about 40% were expanded blastocysts, and a few were early or hatched blastocysts. These data are in agreement with the average developmental rates for IVP. The addition of IGF-I did not increase significantly the cleavage rate or the blastocyst yield. However, the rate of late morulae/blastocysts was significantly increased (P = 0.03) after IGF-I addition to IVC medium but not to IVM medium. These results differ from our preliminary results, in which IGF-I added to IVM medium increased blastocyst yield [26]. This discrepancy may be caused by a difference in slaughterhouse material. In the present study, the ovaries were delivered from distant slaughterhouses, whereas in the previous study oocyte aspiration was performed sooner. The season, which was different during the experimental part of these 2 studies, also can influence the ability of bovine oocytes to develop to the blastocyst stage [36]. Cleavage and blastocyst rate are semiquantitative parameters based on morphological criteria; thus, they are rather subjective and may not give accurate information about embryo viability.
Apoptosis may more likely occur because of suboptimal IVP conditions [14]. Our results confirm those of previous studies [15, 37] showing that bovine IVP blastocyst cells undergo apoptosis. Almost 87% of the blastocysts of the present study showed a positive TUNEL reaction in 1 or more blastomeres. The TUNEL index in blastocysts obtained using the standard IVP protocol (0/0) ranged from 0% to 29%, with an average of 5%. These data are comparable to previous results [15] on bovine IVP embryos cultured in SOFaa without inositol. An apototic index of about 9.5% was obtained by TUNEL on Day 8 blastocysts matured in TCM 199 medium and cultured in SOF medium [37].
In some cases, a cell with fragmented morphology can be TUNEL negative [15]; conversely, not all TUNEL-positive nuclei are in the process of programmed cell death [38]. Therefore, we cannot exclude the influence of these factors on our results. In our study, only green fluorescent cells were counted as TUNEL positive.
The apoptotic index of the blastocysts was not influenced by the addition of IGF-I to the IVM medium. This finding supports those of a previous study [37], where epidermal growth factor added to the IVM medium did not significantly influence the number of apoptotic cells detected by TUNEL, although the blastocyst yield increased in a dose-dependent manner. However, the addition of IGF-I to the IVC medium in the present study reduced the TUNEL index by half. This result indicates that the cleavage period is more crucial for apoptotic development than is oocyte maturation, although IGF-I receptors are present at all stages of maturation and embryo development [8]. IGF-I-induced reduction in the apoptosis index was due to a decrease in the number of apoptotic cells per embryo, but the number of blastocysts with apoptotic cells was not significantly lower after addition of IGF-I.
In most of our control blastocysts, TUNEL-positive cells were in the ICM area or in the ICM/TE area. Only in a few embryos TUNEL-positive cells were exclusively in the TE area. This finding is in agreement with those in previous reports on bovine [14, 15, 24], human [10], and mouse [39] blastocysts. Moreover, IGF-I supplementation of the IVC medium increased the proportion of embryos that had apoptotic cells only in the ICM area. This finding suggests that 1) the TE is the first target of the antiapoptotic effect of IGF-I or 2) IGF-I eliminates apoptotic cells mostly from the TE.
The total cell number did not change after addition of IGF-I to the IVM medium. However, in the IVC medium IGF-I increased significantly the total cell number of the embryos. The decrease in the TUNEL index after the addition of IGF-I to the IVC medium could, at least partially, be explained by the increase in total cell number.
Proliferating cells can be identified in different tissues using specific monoclonal antibodies raised for cyclin/PCNA [40]. PCNA-positive nuclei can be observed during the G1, S, and G2/M phases of the cell cycle [40, 41] but not during the G0 phase [42]. In our study, metaphase nuclei and metaphase chromosome plates did not show staining with PCNA but did incorporate PI. This finding is in agreement with the observation in Drosophila embryos that PCNA dissociates from chromosomes at metaphase [43] and then in later mitotic phases is transported from the cytoplasm into nucleus, where it participates in the formation of the active DNA replication enzyme complexes.
In the present study, we successfully used simultaneous staining and detection of TUNEL-positive and PCNA-positive cells in the same specimen (Fig. 1, A–F). Similar TUNEL indices, obtained by counting of the apoptotic and PI-positive nuclei either from optical sections or from overlay stereoimages, confirm the validity of our immunodetection system using restaining for PI and rescanning of embryos first analyzed for TUNEL/PCNA. Previously, we reported that PCNA can serve as a viability marker of vitrified embryos [20], and we investigated the effect of IGF-I on PCNA in IVP blastocysts [26]. In the present study, about 80% of cells in the control blastocysts were PCNA positive. The rate of PCNA-positive cells increased up to 90% with IGF-I supplementation.
In 60%–66% of all embryos studied, one third of TUNEL-positive cells also showed PCNA signal. This colocalization of PCNA immunoreactivity and TUNEL either in the embryos or in other reproductive tissues has not been reported previously, perhaps because TUNEL and PCNA reactions have not been detected simultaneously on the same specimen. DNA fragmentation is an early stage of apoptotis [10]. Probably, as a response to these nuclear changes, the DNA repair system, including the activation of DNA polymerase delta and PCNA, is activated. Our hypothesis is supported by the earlier report of Kanoh and coworkers [44], who observed the TUNEL/PCNA colocalization in biopsies of human hearts using mirror sections of apoptotic myocytes. Moreover, PCNA positivity was observed in almost 100% of TUNEL-positive cells. On the basis of the TUNEL assay at the electron microscopic level in combination with PCNA and Ki-67 immunocytochemistry, Kanoh and coworkers concluded that PCNA positivity in the TUNEL-positive myocytes means that the cells are undergoing DNA repair rather than S-phase DNA synthesis [44]. However, several researchers have warned about misinterpretation of TUNEL results; DNA strands may break during fixation and specimen preparation [10]. Although it is important to verify apoptosis using other morphological markers, such as nuclear morphology, morphological features of apoptosis are not always associated with DNA fragmentation [45]. Furthermore, TUNEL-positive nuclei are sometimes observed in the absence of morphological features of apoptosis [46]. For more complete evidence of apoptosis, an ultrastructural analysis in addition to TUNEL and nuclear morphology at the light microscopic level may be of value [44; 46].
Day 6.5 blastocysts in our IVP system showed about 5% TUNEL-positive apoptotic cells and almost 80% PCNA-positive cells. The addition of IGF-I to IVC medium, but not to IVM medium, reduced the percentage of TUNEL-positive cells by almost half and significantly increased the total cell number of blastocysts. The reduction in the TUNEL index might be explained partially by the decrease in the number of apoptotic cells per embryo and partially by the increase in the total cell number of blastocysts. The increase in the PCNA reaction after the addition of IGF-I to either IVM medium or IVC medium was not significant. Colocalization of TUNEL and PCNA signals indirectly suggests an activation of the DNA repair process, involving PCNA as a component of DNA polymerase delta in TUNEL-positive nuclei. The present observations indicate that for the prevention of apoptosis in bovine IVP embryos, the growth factor requirement is more critical during embryo culture than during oocyte maturation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Ministry of Agriculture and Forestry, Finland, and by Tekes, the National Technology Agency, Finland. 
2 Correspondence: A.V. Makarevich, Research Institute of Animal Production, Hlohovska 2, SK-949 92 Nitra, Slovakia. FAX: 421 37 6546 361; makarev{at}vuzv.sk
Accepted: September 17, 2001.
Received: April 23, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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