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Biology of Reproduction 65, 52-59 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Ratio of Proliferating Cell Nuclear Antigen-Positive Nuclei to Total Cell Number Is Higher in Day 7 Than in Day 8 Vitrified In Vitro-Produced Bovine Embryos1

M. Markkula2,a, M. Rätya, L. Jauhiainena, J. Parankob, J. Raulaa, and A. Makarevicha,c

a Agricultural Research Centre of Finland, Animal Production Research, FIN-31600 Jokioinen, Finland b University of Turku, Department of Biology, FIN-20014 Turku, Finland c Research Institute of Animal Production, SK-94992 Nitra, Slovak Republic

ABSTRACT

The aim of the present study was to find a reliable functional criterion for the evaluation of the proliferation potential of bovine in vitro-produced embryos. We used immunocytochemical detection of proliferating cell nuclear antigen (PCNA) combined with propidium iodide (PI) staining and subsequent confocal laser scanning microscopy together with routine morphological evaluation under a stereomicroscope to study fresh Day 7, 8, and 9, and cryopreserved Day 7 and 8 embryos. The ratio of PCNA/PI-positive nuclei was equal in fresh Day 7 and Day 8 embryos and significantly lower in Day 9 embryos. In general, Day 7 embryos tolerated the cryopreservation treatments better than Day 8 embryos. Vitrification in normal straws was especially detrimental to Day 8 embryos. In fresh Day 7 and 8 embryos, the PCNA results were in agreement with stereomicroscopic evaluation. However, in Day 9 fresh and in Day 7 and 8 treated embryos, the missing PCNA revealed disorders that were not observed under morphological evaluation. PCNA immunocytochemistry is an effective method to obtain information about the functional state of nuclei. The ratio of PCNA-positive nuclei can provide more information and numerical data about the developmental potential of bovine embryos after cryopreservation.

conceptus, embryo development

INTRODUCTION

In vitro embryo production of mammalian, especially cattle embryos, has been intensified remarkably during recent years [1]. Ultrasound-guided follicular aspiration of oocytes [1, 2], subsequent in vitro production (IVP) and cryopreservation of the embryos are pivotal tools in facilitating intensive commercial breeding programs in cattle. The main obstacle in this strategy is, however, different development of in vitro-produced bovine embryos from that of their in vivo counterparts [3]. Particularly, the cryopreservation of IVP embryos has been reported to be less successful [4, 5]. General improvements in IVP procedures resulting in higher percentages of good quality and early developing blastocysts can also increase the viability of the IVP embryos after cryopreservation [6, 7].

Vitrification is a relatively new and to date the most suitable method for cryopreservation of IVP embryos [8–10]. Generally, embryos are vitrified at blastocyst or expanded blastocyst stage and cultured after warming for evaluation of cryopreservation technique. However, visual estimation of embryos does not reveal their developmental potential. No single method for an accurate viability test of preimplantation embryos is yet available [11].

The goal of present study was to find a reliable functional criterion for estimation of the viability of in vitro-produced bovine embryos and to compare the proportion of proliferating cells in the embryos that have undergone vitrification procedures. Proliferating cells have been identified in developing and tumorous tissues [12], in Xenopus embryos [13] and fertilized mouse oocytes [14] using a specific monoclonal antibody raised for cyclin/proliferating cell nuclear antigen (PCNA) [15, 16]. PCNA is an evolutionarily highly conserved 36-kDa acidic nuclear protein that is essential for DNA synthesis [17]. It functions as an auxiliary protein for DNA-polymerase delta [18–21] both in the S-phase [22] and in DNA repair [23]. PCNA-positive nuclei can be observed during the G1-, S-, and G2/M-phases of the cell cycle [16, 24] but not in G0 [22].

In this study, we have applied PCNA immunocytochemistry, in combination with laser confocal microscopy, for the analysis of bovine IVP embryos cryopreserved by two methods, vitrification in normal straw [9] and in open-pulled-straw (OPS) [25], to evaluate whether embryos reaching blastocyst stage on Days 7 and 8 are equally suitable for cryopreservation by these methods.

MATERIALS AND METHODS

Unless otherwise specified, chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), and those used for embryo production were of embryo-tested quality.

In Vitro Embryo Production

Ovaries were collected from slaughtered cattle of mixed breeds. Follicular fluid containing the oocytes was aspirated and transported to the laboratory within 6 h at 36 ± 2°C. All cumulus-oocyte complexes, except the overmatured, were used for IVP. They were washed twice in Hepes-buffered Talp (125.4 mM NaCl, 3.16 mM KCl, 2.0 mM NaHCO3, 0.35 mM NaHPO4, 10.0 mM Na-lactate, 2.0 mM CaCl2·2H2O, 0.5 mM MgCl·6H2O, 10.0 mM Hepes), and once in maturation medium (TCM-199 with Glutamax; Gibco, Grand Island, NY), supplemented with FSH (2 µg/ml Ovagen; 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 10% fetal calf serum (FCS; Gibco BRL, Auckland, New Zealand). Fifty oocytes were matured in 500 µl of maturation medium by incubation under mineral oil at 39°C and 5% CO2 in humidified air for 24 h.

Frozen-thawed and washed sperm from a single Ayrshire bull of a proven in vitro fertility was used for fertilization. Spermatozoa (1.5 x 106/ml) and 100–125 cumulus-oocyte complexes were incubated in 500 µl of fertilization medium for 20 h at 39°C under 5% CO2 in humidified air.

Presumptive zygotes were vortexed for 90 sec in Hepes-Talp to remove the cumulus cells. The denuded zygotes were washed once in Hepes-Talp and twice in culture medium, SOFaaci [26] supplemented with 5% FCS, placed in groups of 50 in a 35-µl drop of SOFaaci under mineral oil and incubated in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 at +39°C.

Experimental Groups

On Days 7, 8, and 9 (Day 0 = day of insemination), embryos with a defined blastocoele were removed from the original culture into Hepes-Talp and evaluated according to their developmental stage: early blastocyst (eBl), blastocyst (Bl), expanded blastocyst (xBl), hatching or hatched blastocyst (hBl). According to embryo quality, they were further evaluated by one person as belonging to classes I–II, III, and dead. Classes were defined according to the color (light, I—dark, III), compaction of the ICM (compact I—dispersed III) and integrity of the trophectoderm (even I—disintegrated III). The embryos were then divided into six experimental groups: 1) CC, fresh control embryos that were placed in groups of one to three into a fresh SOFaaci drop for further culture; 2) FI, immunocytochemistry was performed immediately; 3) VM, embryos were taken through the vitrification solutions [9] and loaded into normal 0.25-ml straws, washed, but not exposed to liquid nitrogen; 4) OM, embryos were put through the OPS vitrification solutions and loaded into smaller OPS straws [25], washed, but not exposed to liquid nitrogen; 5) V, complete vitrification procedure [9] including loading into a normal 0.25-ml straw, cooling, and warming; and 6) O, complete OPS procedure including loading into a special OPS straw, cooling, warming, and washing [25]. Experimental embryos VM, OM, V, and O were placed after the treatments in groups of one to three embryos into a fresh drop of SOFaaci for further culture overnight as above. After the culture, the embryos (except those of group FI) were reevaluated according to their morphology under a stereomicroscope and finally subjected to immunocytochemistry.

Vitrification in Normal and Open-Pulled Straws

The vitrification and OPS procedures were performed as described earlier [9, 25]. In short, for vitrification (V), the embryos were first incubated for 5 min in 500 µl holding medium, and then in the same volume of 50% vitrification solution consisting of 12.5% ethylene glycol, 12.5% dimethylsulfoxide, and 75% holding medium for 60 sec at 23 ± 2°C. Embryos were then incubated for 20 sec in three droplets of 100% vitrification solution containing 25% ethylene glycol, 25% dimethylsulfoxide, and 50% holding medium at +4°C. Normal 250-µl French straws (IMV, L'Aigle, France) were loaded with 180 µl of +4°C holding medium and then with three 5-µl droplets of 100% vitrification solution. All droplets were separated from each other and the holding medium by air bubbles. The middle droplet contained two to four embryos/straw. Straws were sealed immediately with a cap, placed horizontally in liquid nitrogen vapor for 2 min, and finally immersed in liquid nitrogen. For warming, the straws were immersed horizontally in a waterbath at +22°C for 8 sec, shaken three times, and incubated horizontally for a further 5–30 min in the same waterbath. Embryos were expelled and washed three times in holding medium before the culture.

For OPS, all media and dishes were warmed to +39°C. One to four embryos were incubated twice in 800 µl of holding medium with 20% FCS for 1 min, and then for 3 min in 800 µl of vitrification solution I containing 7.5% ethylene glycol, 7.5% dimethylsulfoxide, and 85% holding medium. Embryos were transferred into 20 µl of vitrification solution II containing 16.5% ethylene glycol, 16.5% dimethylsulfoxide, and 67% holding medium with 0.5 M sucrose and quickly taken with about 1 µl of vitrification solution II into OPS. The straws were immersed into liquid nitrogen first horizontally then turning vertical. For warming, the thinner end of the straw with the vitrified embryos was immersed in holding medium with 20% sucrose medium [25], and emptied. After 1 min, embryos were transferred into vitrification medium containing 11% sucrose medium for 5 min. After one 5-min and a second short wash in holding medium with 20% FCS, embryos were placed onto culture dishes.

PCNA Immunocytochemistry and Propidium Iodode Staining

Embryos, one to three in a group, were washed three times, 5 min each, in a 50-µl drop of PBS supplemented with polyvinylpyrrolidone (PBS-PVP, 4 mg/ml). This was followed by fixation at room temperature for 5 min in neutral buffered formalin (3.7%), washing, and postfixation for 10 min in 70% ethanol. For membrane permeabilization, embryos were incubated in 0.1% Triton X-100 in PBS for 5 min. Nonspecific binding of antibodies was suppressed by incubation in a blocking solution (0.1% BSA in PBS). Thereafter, embryos were transferred for 1 h into a drop of monoclonal anti-PCNA antibody (Dako, clone PC-10; Dako A/S, Glostrup, Denmark) diluted 1:50 in blocking solution. Specimens were washed three times for 5 min each time in PBS-PVP. For the visualization of PCNA-labeled nuclei, embryos were incubated for 30–40 min in the presence of fluorescein isothiocyanate (FITC)-labeled anti-mouse immunoglobulins (Dako A/S) at a dilution of 1:50 in blocking solution. No reaction was observed when the primary antibody was omitted.

For the visualization of all nuclei, fluorescent propidium iodide (PI) at the final concentration of 200 µg/ml was added to the secondary antibody solution and thereafter washed three times for 5 min each time in PBS-PVP.

The washed embryos were placed onto a coverslip and immediately mounted in 6 µl of 1% agar dissolved in the solution containing 100 mg 1,4-diazobicyclo(2.2.2)octane and 50% glycerol in PBS. Before use, the agar was melted in a microwave oven. The coverslip was set on an object glass to rest on supports made by drops of nail polish. Finally, glycerol diluted with PBS (2:1) was applied under the coverslip to fill the remaining spaces between the coverslip and the object glass. The attachment of the coverslip was secured with nail polish. Specimens were stored at -20°C until evaluation.

Laser Confocal Microscopy and Counting of Nuclei

Whole-mount embryos were studied with a Leica TCS NT confocal microscope using simultaneous detection for PCNA and PI. The number of stained nuclei was counted on each optical section image, each representing a 0.8-µm slice of the embryo. Most likely, the same nuclei were counted in several sections.

Statistical Analyses

The Kruskal-Wallis ANOVA with a simulation-based approach [27] was used to analyze the statistical differences in the developmental stage of the embryos before and after the treatments. Day 7 and 8 embryos were analyzed separately.

The ratio of PCNA/PI-positive nuclei was analyzed using ANOVA including the age of the embryo, the treatment and their interactions as fixed effects, and residual as a normally distributed random effect. Assumptions of the models were checked using graphical methods: box-plot for normality of errors and plots of residuals for constancy of error variance [28]. Normal distribution of the data was achieved after the following transformation: square root of 100% minus PCNA/PI ratio. However, all the presented estimates were transformed to the original scale. All statistical analyses were performed using Statistical Analysis Systems software.

RESULTS

Morphological Characteristics of Embryos

A total of 1954 oocytes were used for embryo production. The average cleavage rate was 81% and total blastocyst yield was 21%.

Embryos selected for the control (CC) and different treatments (OM, VM, O, and V) were at the same developmental stage. In the CC group, hatching rate was higher among embryos that developed a blastocoel on Day 7 (14/16, 88%) than on Day 8 (9/17, 53%) or Day 9 (4/16, 25%).

After the treatments and subsequent individual culture overnight, there were statistically significant differences in the developmental stages in Day 8 but not in Day 7 embryos (P < 0.0001 and P = 0.16, respectively) (Table 1). Among the Day 7 embryos, the number of dead embryos was increased only after vitrification (V) (P < 0.06). The other treatments had no effect on the developmental stage (Table 1) or quality (Table 2) of the embryos, when evaluated on the basis of their morphological appearance under the stereomicroscope. Day 8 embryos did not show developmental retardation in their development or signs of degeneration after exposure to the vitrification (VM, P = 0.55) or the OPS (OM, P = 0.95) protocol when compared to fresh control embryos. Only after subsequent cooling and warming did Day 8 embryos degenerate and remain retarded in their development (V, P < 0.0001 and O, P = 0.04) when compared to fresh control embryos. After vitrification (V) in normal-sized straws, the developmental stage of the Day 8 embryos was lower (P = 0.06) than after OPS vitrification (O). When groups of Day 7 and Day 8 embryos were compared, the developmental stage of the Day 8 embryos was significantly lower (P < 0.005) only after vitrification in normal-sized straws (V).


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  		TABLE 1. Developmental stage of the Day 7 and Day 8 embryos before and after treatments and subsequent culture overnight


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  		TABLE 2. Quality of embryos after treatments and individual culture overnight

After cooling, warming, and overnight recovery in culture, more embryos were classified as totally degenerated after vitrification in normal-sized straws (V) than after OPS in thinner straws (Table 2). Compared to the Day 7 embryos, Day 8 embryos were more sensitive to the treatments used in the present study. This was particularly prominent after vitrification in 0.25-ml straws (V).

Only a few embryos developed to blastocyst stage on Day 9. Therefore, they could not be divided into treatment groups but only subjected to staining.

Proliferation Potential

Most commonly, PCNA-positive nuclei were well defined, and the negative nuclei were found scattered in the trophectoderm cells and in the inner cell mass of fresh Day 7 (Fig. 1, A–C) and Day 8 embryos (Fig. 1, D–F). Fresh Day 9 embryos demonstrated very faint PCNA staining (Fig. 1, G and I) and diffuse nuclear PI staining (Fig. 1, H and I). Fresh control Day 7 and 8 embryos showed an equal percentage (91–88%) of PCNA-positive nuclei, but that of Day 9 embryos was significantly lower (Table 3). The cell number was very variable in all age groups, irrespective of the developmental stage or quality classification of the embryos, as shown by nuclear staining in Figures 1–3. However, their morphological appearance and developmental stage was the same (Fig. 1, J–L).



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  		FIG. 1. Representative images of confocal laser scanning microscopy of fresh Day 7 (A–C), Day 8 (D–F), and Day 9 (G–I) bovine embryos. Nuclear staining after immunocytochemistry with anti-PCNA-antibody and FITC-labeled secondary antibody is fainter and the number of PCNA-positive cells is lower in Day 9 embryo (D) than in Day 7 (A) or Day 8 (D) embryos. The total number of nuclei per embryo was variable in all age groups as shown here in B, E, and H with PI staining. The ratio of PCNA/PI-labeled nuclei was equal in Day 7 and 8 embryos, but it was significantly lower in Day 9 embryos, although their developmental stage under stereomicroscopic evaluation (Day 7, J, Day 8, H, and Day 9, L) was the same, expanded blastocyst. Bars = 100 µm


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  		TABLE 3. Percentage of PCNA-positive nuclei in the embryos of different age after the treatments

Among the Day 7 embryos, both actual cryopreservation methods, vitrification (V) and OPS (O), and subsequent warming significantly reduced the percentage of PCNA-positive nuclei compared to the fresh control embryos (P < 0.005 and P = 0.01, respectively). The exposure to cryomedia and loading into straws without actual contact with liquid nitrogen (VM and OM) did not significantly reduce the percentage of PCNA-positive nuclei in Day 7 embryos (Table 3). Figure 2 shows Day 7 embryos that have been subjected to treatments OM, VM, O, and V. Panels A, D, G, and J show the PCNA staining, panels B, E, H, and K the PI staining, and panels C, F, I, and L combined PCNA/PI staining after the treatments OM (panels A–C), VM (panels D–F), O (panels G–I), and V (panels J–L).



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  		FIG. 2. Confocal laser scanning microscopy of Day 7 bovine embryos after the treatments, recovery overnight in culture, and immunocytochemistry with anti-PCNA-antibody and staining with PI. Panels A–C show an embryo that was put through the OPS vitrification solutions and loaded into the OPS straws without exposure to liquid nitrogen but with subsequent unloading and washing (OM). Panels D–F represent an embryo that was exposed to the vitrification solutions and loading into a normal 0.25-ml straw and subsequent washing but without exposure to liquid nitrogen (VM). Panels G–I show an embryo that was subjected to the complete OPS procedure (O). Panels J–L show an embryo that was subjected to the complete vitrification procedure (V). Bars = 100 µm

Among the Day 8 embryos, the ratio of PCNA-positive nuclei was reduced in OM (P < 0.005), V (P < 0.0001), and O (P < 0.005), but not in VM experimental embryos when compared to the controls (FI) (Table 3). Figure 3 shows examples of confocal section image overlays of Day 8 embryos after the four treatments. Panels A, D, G, and J show the PCNA staining, panels B, E, H, and K the PI staining, and panels C, F, I, and L combined PCNA/PI staining after the treatments OM (panels A–C), VM (panels D–F), O (panels G–I), and V (panels J–L).



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  		FIG. 3. Confocal laser scanning microscopy of Day 8 bovine embryos after the treatments, recovery overnight in culture, and immunocytochemistry with anti-PCNA-antibody and staining with PI. Panels A–C show an embryo that was put through the OPS vitrification solutions and loaded into the OPS straws without exposure to liquid nitrogen but with subsequent unloading and washing (OM). Panels D–F represent an embryo that was exposed to the vitrification solutions and loading into a normal 0.25-ml straw and subsequent washing but without exposure to liquid nitrogen (VM). Panels G–I show an embryo that was subjected to the complete OPS procedure (O). Panels J–L show an embryo that was subjected to the complete vitrification procedure (V). Bars = 100 µm

Among Day 7 embryos, vitrification in normal straws (V) and the OPS method were equally successful when measured as the ratio of PCNA-positive nuclei, whereas Day 8 embryos that underwent vitrification (V) revealed a lower percentage of PCNA-positive nuclei, compared to their counterparts after the OPS (O) method (P = 0.04) (Table 3). Furthermore, Day 8 embryos had significantly less PCNA-positive nuclei than Day 7 embryos after the OPS protocol (OM, P < 0.0001) and vitrification (V, P < 0.005).

DISCUSSION

Proper evaluation of the viability of in vitro-produced bovine embryos is one of the critical factors in intensive embryo production. Visual evaluation is not sufficient for a reliable estimation of the in vivo viability of the in vitro-derived embryos. In particular, when applying new methodological approaches, morphological classification of the embryos needs to be validated. Earlier studies of the vitrified embryos have revealed that even though, under stereomicroscopic evaluation, the surviving embryos may not show any sign of previous injury, unexpectedly extensive damage can be detected at the ultrastructural level [29]. Electron microscopy and embryo transfer can provide reliable information about structural damage and in vivo viability, respectively, but may not be practically available in all laboratories. In the present study, we have used immunocytochemical detection of PCNA as a marker for the proliferation potential of fresh and cryopreserved IVP bovine embryos. Embryos were fixed and processed for immunoreactions in drops and attached onto slides only at the end of the protocol. The method is fast and enables the staining of proliferating nuclei simultaneously in several embryos.

In our study, morphological evaluation of fresh Day 7 and 8 embryos under the stereomicroscope and by PCNA/PI staining gave similar results. Before the treatments, both Day 7 and Day 8 embryos had an equal ratio of proliferating nuclei and they were at the same developmental stage. The total cell number of the embryos could not be counted but only estimated from the optical section images, because some nuclei were obviously present in several sections. However, it became evident that the number of nuclei in the embryos varied, being remarkably low in some blastocysts. This was noted in all groups regardless of their age. These differencies could not be observed under the stereomicroscope. Embryos that reached the blastocyst stage more slowly, on Day 8, were not only of lower quality but also more sensitive to the treatments compared to the Day 7 embryos. This could be confirmed by their morphological features, hatching rate, and the PCNA/PI ratio. This observation is in agreement with the previous report on Day 7 and Day 8 embryos [30]. It further confirms the data indicating significantly lower pregnancy rates obtained by the Day 8 blastocysts [31].

Morphologically, the quality of Day 7 embryos was decreased after the exposure to vitrification solutions and even more affected after complete vitrification, including exposure to liquid nitrogen. This observation was also confirmed by the functional criteria, detection of the proportion of PCNA-positive nuclei. Of the two cryopreservation procedures, the OPS protocol without (OM) or with (O) immersion in liquid nitrogen was tolerated better by Day 7 embryos than was the vitrification in the normal straw.

Day 8 embryos tolerated equally the two cryopreservation protocols when evaluated on a morphological basis. However, the percentage of PCNA-positive nuclei was significantly lower after the OPS protocol (OM). Both cryopreservation methods damaged the Day 8 embryos significantly, when evaluated on a morphological basis and by PCNA immunocytochemistry. However, in both methods used, vitrification in normal straws was significantly more damaging for the older blastocysts. This is in agreement with our observation that Day 9 fresh blastocysts (without any treatments) exhibited a significantly lower proportion of PCNA-positive nuclei than Day 7 or Day 8 fresh embryos (Table 3, Fig. 1), although under the stereomicroscope the differences were not observed.

PCNA immunoreactivity can be demonstrated after fixation in a wide range of fixatives [32]. Formalin fixation may slightly increase the number of PCNA-immunopositive nuclei. This may be due to the staining of the PCNA associated with replication sites and the nucleoplasmic PCNA present at low levels in cells that are actually not replicating [33]. In our preliminary study with bovine blastocyst stage embryos, the staining pattern both after methanol-acetone and formalin fixation was similar (data not shown) under confocal microscopy. In this study, formalin fixation was used.

It may be that some apoptotic nuclei were regarded as PCNA positive, although they actually were no longer in the cell cycle, because the half-life of PCNA is approximately 20 h [33]. PCNA is considered to be undetectable in long-term quiescent cells by immunocytochemical methods, even though it may be present at low levels, i.e., 10% of that of cycling cells, as demonstrated by molecular biological techniques [32]. In a preliminary study on the preservation of PCNA immunoreactivity, we stopped the development of Day 8 embryos with sodium azide, and detected the proportion of PCNA-positive nuclei as in the present study. Even after only 12 h of sodium azide treatment, the proportion of PCNA-positive nuclei was significantly (P < 0.05) reduced from 82.8% (control group) to 62.3%, and after 24 h, it was decreased to 48.6% (P < 0.01) [34]. There is also a highly significant correlation (r = 0.603, P < 0.0001) between PCNA-positive cells and the total cell number of Day 7 embryos (n = 156, data not shown).

In conclusion, our results show that the ratio of PCNA/PI-positive nuclei can provide valuable information and numerical data about the viability of bovine embryos, which may be regarded as a functional criterion for the evaluation of embryo viability after cryopreservation.

ACKNOWLEDGMENTS

The authors are grateful to Tuula-Marjatta Nieminen for technical help in the laboratory and to Elina Laihonen and Outi Kasari for help in preparing the figures.

FOOTNOTES

First decision: 2 November 2000.

1 This work was supported by the Ministry of Agriculture and Forestry, Finland and Tekes, the National Technology Agency, Finland. Back

2 Correspondence. FAX: 358 3 4188 8618; merja.markkula{at}mtt.fi Back

Accepted: February 9, 2001.

Received: August 28, 2000.

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A.V. Makarevich and M. Markkula
Apoptosis and Cell Proliferation Potential of Bovine Embryos Stimulated with Insulin-Like Growth Factor I During In Vitro Maturation and Culture
Biol Reprod, February 1, 2002; 66(2): 386 - 392.
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