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Biology of Reproduction 65, 793-798 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Energy Status of Nonmatured and In Vitro-Matured Domestic Cat Oocytes and of Different Stages of In Vitro-Produced Embryos: Enzymatic Removal of the Zona Pellucida Increases Adenosine Triphosphate Content and Total Cell Number of Blastocysts

Peter Freistedta, Petra Stojkovica, Eckhard Wolfa, and Miodrag Stojkovic1,a

a Department of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, D-85764 Oberschleissheim, Germany

ABSTRACT

In this study, we evaluated the adenosine triphosphate (ATP) content of individual domestic cat oocytes before and after in vitro maturation and of different stages of in vitro-produced embryos. To investigate the effects of assisted-hatching technique on the ATP content and total cell number, the zona pellucida of in vitro-produced blastocysts and expanded blastocysts (recovered 144 h postinsemination [hpi]) was completely removed by pronase treatment. The average (mean ± SEM) ATP content of nonmatured oocytes (3.47 ± 0.18 pmol) was significantly (P < 0.01) higher than that of in vitro-matured oocytes (2.17 ± 0.10 pmol). After in vitro fertilization and culture, the ATP content of two-cell stages (24 hpi) was 1.17 ± 0.08 pmol, which increased to 1.47 ± 0.19 and 1.88 ± 0.32 pmol at the four- (40 hpi) and eight-cell (48 hpi) stages, respectively. The ATP content then decreased to 1.48 ± 0.10 pmol in 16-cell embryos (64 hpi), reaching a minimum of 0.49 ± 0.04 pmol at the morula stage (120 hpi). Blastocysts, expanded blastocysts (both 144 hpi), and hatching blastocysts (192 hpi) revealed ATP levels of 1.05 ± 0.09, 1.79 ± 0.01, and 4.17 ± 0.21 pmol, respectively. After enzymatic removal of the zona pellucida (ERZP) at 144 hpi, ATP content and total cell numbers of blastocysts (4.15 ± 0.37 pmol of ATP, 328.3 ± 48.5 cells) and expanded blastocysts (5.81 ± 0.54 pmol of ATP, 430.1 ± 29.7 cells) analyzed at 192 hpi were significantly (P < 0.001) higher than in their nontreated counterparts (blastocysts: 1.00 ± 0.09 pmol of ATP, 65.3 ± 4.6 cells; expanded blastocysts: 1.79 ± 0.11 pmol of ATP, 121.4 ± 6.5 cells). Our study describes, to our knowledge for the first time, changes in the energy status of domestic cat oocytes before and after maturation and during in vitro development after fertilization. The ERZP markedly increased the ATP content and total cell number of blastocyst stages, suggesting that this technique may improve the quality and viability of in vitro-produced domestic cat embryos.

early development, in vitro fertilization, oocyte development, ovum

INTRODUCTION

Metabolic activity and energy production are necessary features of nuclear and cytoplasmic maturation of oocytes [1], resumption of meiosis of oocytes [1–3], successful cleavage after fertilization [2–6], and hatching and implantation of embryos [7, 8]. The adenosine triphosphate (ATP) content of oocytes and embryos is critical for nucleic acid and protein synthesis [8, 9], and ATP content has been suggested as an indicator for the developmental potential of human [7, 10], mouse [8, 9, 11], and bovine oocytes and embryos [4]. Metabolism of glucose, palmitic acid, glutamine, pyruvate, and lactate supplies the oocyte with the energy required during maturation and for further development. Therefore, metabolic activity has been suggested as an indicator of the developmental potential and vitality of domestic cat oocytes and embryos [12]. The in vitro production of domestic cat embryos is already established [13–23], but the transfer of in vitro-produced domestic cat embryos results in higher embryonic loss than occurs after transfer of in vivo-recovered embryos [24]. Therefore, recognition of the nutritional requirements and improvement of the culture conditions will increase the developmental potential of in vitro-produced cat embryos.

Additionally, after transfer, the release from the zona pellucida is essential for further embryonic development in utero. In domestic cat embryos, zona pellucida dissolution, beginning at the abembryonic pole, occurs due to enzymatic activity of at least the trophoblast [25], and it requires a high amount of energy [26]. Some evidence suggests that changes of the zona pellucida during early embryonic development are quite different under in vitro conditions as compared to the in vivo situation. For instance, in vivo embryos collected at the morula stage showed a gradual and severe thinning of the zona pellucida when cultured in vitro [27]. This phenomenon was pronounced over one pole and associated with a local zona dissolution that allowed the expanded blastocyst to emerge. On the other hand, in embryos collected during earlier stages, zona thinning was not seen, and embryos became entrapped when trying to escape from a small hole formed in the zona pellucida [27, 28]. Inefficient utilization of energy substrates during in vitro culture and/or structural changes of the zona pellucida caused by these conditions may lead to the altered zona dissolution of domestic cat embryos. Studies with mouse, human [29], and bovine [30] embryos indicate that several treatments (i.e., zona drilling, zona slitting, partial zona dissection, zona thinning, and zona digestion) improve the developmental potential of blastocysts cultured in vitro.

To our knowledge, however, no study has evaluated the effects of enzymatic removal of the zona pellucida (ERZP) on the viability and quality of in vitro-produced domestic cat embryos. Therefore, we measured the ATP content of individual nonmatured and in vitro-matured oocytes and of different stages of in vitro-produced domestic cat embryos. Furthermore, we studied the effect of assisted hatching (i.e., zona digestion) on ATP content and total cell numbers of blastocysts and expanded blastocysts. Both ATP content [4, 7–10] and total cell number [31, 32] have been suggested as parameters of embryo vitality.

MATERIALS AND METHODS

Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Collection and Transport of Gonads

Female and male gonads were recovered from domestic cats that were kept under noncontrolled housing conditions. The gonads were collected at local veterinary clinics weekly from March to September 2000. Pairs of ovaries and testes were transferred immediately after excision into Dulbecco phosphate-buffered saline (PBS; pH 7.4, containing 100 IU/ml of penicillin and 100 IU/ml of streptomycin sulfate; Biochrom KG, Berlin, Germany).

Oocyte Recovery and In Vitro Maturation

Ovaries from each cat were placed into a 60-mm Petri dish containing TCM199 (Gibco BRL, Paisley, Scotland) supplemented with 3 mg/ml of BSA, 0.6 mg/ml of potassium lactate, 3 mg/ml of NaHCO3, 1.4 mg/ml of Hepes, 0.25 mg/ml of pyruvate, 0.1 mg/ml of cysteine, and 0.055 mg/ml of gentamicin. The cumulus-oocyte complexes (COCs) from antral follicles were released by slicing the ovarian cortex using a scalpel blade. Only COCs with a uniformly dark, finely granulated, or slightly less darkly pigmented ooplasm and a complete, compact cumulus cell investment of at least two cell layers were selected and washed three times in the same medium. The COCs were cultured in 400 µl of maturation medium composed of the above-described medium supplemented with 0.02 IU/ml of bovine FSH and 0.01 IU/ml of bovine LH (Sioux Biochemical, Sioux Center, IA) for 24–28 h at 39°C in an atmosphere of 5% CO2 in air and maximum humidity.

Sperm Recovery and In Vitro Fertilization

The cauda epididymidis and the first 1 cm of the ductus deferens were dissected from the testis and placed in a 35-mm Petri dish containing 800 µl of Hepes-Tyrode albumin lactate pyruvate (TALP) medium. Epididymal sperm were released by mincing using a scalpel blade. Tissue debris was removed, and the remaining sperm suspension was diluted 1:2 (v/v) with a commercial sperm extender based on Tris-buffer and egg yolk (Biladyl A; Minitüb, Tiefenbach, Germany). Sperm were used for insemination either on the day of recovery or stored at 4°C for as long as 5 days in the Tris-buffered egg yolk medium. Before use, they were subjected to a modified swim-up treatment by gently layering 100 µl of sperm suspension under 200 µl of Hepes-TALP medium [33], followed by a 1-h incubation at 39°C. The clear supernatant was aspirated, sperm motility estimated, and sperm concentration determined using a hemocytometer. In vitro fertilization (IVF) was performed by adding 105 motile spermatozoa to the in vitro-matured oocytes in 400 µl of IVF medium (TALP solution containing 6 mg/ml of BSA and 10 µg/ml of heparin) and incubating for 20–22 h at 39°C in 5% CO2 in air.

In Vitro Culture

Presumptive zygotes were washed three times and cultured in 400 µl of synthetic oviduct fluid (SOF) medium [34] supplemented with 2% (v/v) Eagle basal medium amino acids, 1% (v/v) nonessential amino acids (Gibco BRL, Paisley, Scotland), and 10% (v/v) estrous cow serum (SOF/ECS). At 36 h postinsemination (hpi), putative zygotes were washed three times in culture medium to remove cumulus cells and were then examined for cleavage. Embryos were transferred into 400 µl of fresh SOF/ECS under equilibrated paraffin oil and cultured at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2.

Enzymatic Removal of the Zona Pellucida

The zona pellucida of blastocysts and expanded blastocysts was completely digested at 144 hpi with 0.5% pronase in PBS. The embryos were washed three times and cultured for the next 2 days in SOF/ECS.

Measurement of the ATP Content

The cumulus cells of nonmatured and matured oocytes were removed by hyaluronidase (1 mg/ml) in PBS supplemented with 3 mg/ml of BSA and by vortexing (4 min). After 24 h of in vitro maturation (IVM), oocytes were checked for presence of the first polar body. The ATP content of samples, that is, completely denuded nonmatured and matured oocytes with the presence of the first polar body, 2-cell (24 hpi), 4-cell (40 hpi), 8-cell (48 hpi), 16-cell (64 hpi), morula (120 hpi), and blastocyst and expanded blastocyst stages (144 hpi), was measured. In addition, at 192 hpi, the ATP content of blastocysts, expanded blastocysts, and hatching blastocysts with or without ERZP treatment was measured (Fig. 1). The ATP content of each oocyte or embryo was measured with a commercial assay based on the luciferin-luciferase reaction (Bioluminescent Somatic Cell Assay Kit, FL-ASC) as previously described [35] with some minor modifications. Briefly, samples were rinsed three times in PBS supplemented with 3 mg/ml of BSA, three times in sample buffer (99.0 mM NaCl, 3.1 mM KCl, 0.35 mM NaH2PO4, 21.6 mM Na-lactate, 10.0 mM Hepes, 2.0 mM CaCl2, 1.1 mM MgCl2, 25.0 mM NaHCO3, 1.0 mM Na-pyruvate, 0.1 mg/ml of gentamicin, and 6.3 mg/ml of BSA), and transferred individually in 50 µl of sample buffer into plastic tubes on ice water. Then, 100 µl of ice-cold somatic cell reagent (FL-SAR) were added to all tubes, which were incubated for 5 min on ice water. Subsequently, 100 µl of ice-cold assay mix (diluted 1:25 with ATP Assay Mix Dilution Buffer, FL-AAB) were added, and the tubes were kept for 5 min at room temperature in the dark. The ATP content was measured using a luminometer (Bioluminat Junior; Berthold, Wildbad, Germany) with high sensitivity (0.01 pmol). A seven-point standard curve (0–6 pmol/tube) was routinely included in each assay. The ATP content was determined from the formula for the standard curve (i.e., linear regression).



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  		FIG. 1. Time schedule for the recovery and ATP measurements of nonmatured and in vitro-matured oocytes and different stages of embryos, including blastocyst and expanded blastocyst stages. The most-advanced stages were treated or not treated by enzymatic removal of the zona pellucida (ERZP). IVC, In vitro culture

Total Cell Numbers of Embryos

Nontreated and pronase-treated blastocysts and expanded blastocysts were stained at 192 hpi with 2 µg/ml of Hoechst 33342. The cell numbers of embryos were counted using epifluorescence microscopy (Axiovert 135; Zeiss, Jena, Germany).

Statistical Analysis

All experiments were repeated at least three times, except for one experiment in which the effect of individual donors on the ATP content of recovered nonmatured and matured oocytes was investigated. When a significant (P < 0.05) F-statistic was found, the ATP contents of recovered oocytes and embryos were compared using ANOVA followed by Bonferroni post-hoc tests. Total cell numbers of embryos were compared using Mann-Whitney U-tests. The overall correlation between ATP content and total cell number of embryos recovered at 192 hpi was evaluated by calculating the Spearman-Rho correlation coefficient. Effects of assisted-hatching treatment on the ATP content per blastocyst or single embryonic cell were evaluated using Student t-test. A value of P < 0.05 was considered to be statistically significant.

RESULTS

ATP Content of Oocytes Before and After IVM

The ATP content of nonmatured oocytes was significantly (P < 0.05) higher than that of in vitro-matured oocytes (Fig. 2). Nonmatured oocytes contained 3.47 ± 0.18 pmol of ATP, and oocytes that extruded the first polar body 24 h after IVM had 2.72 ± 0.10 pmol of ATP.



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  		FIG. 2. The ATP content of nonmatured (n = 51) and in vitro-matured (n = 34) domestic cat oocytes. Data are presented as the mean (bars) and SEM (error bars). Means were compared using ANOVA followed by Bonferroni post-hoc test. *P < 0.05

To evaluate the effect of donor on the ATP content of nonmatured and matured oocytes, we performed a small experiment in which oocytes from three donors (cats 1, 2, and 3) were treated and measured separately. A significant effect of donor animal on the ATP content of oocytes was seen both before (Fig. 3A) and after (Fig. 3B) IVM. However, for all donors, the ATP content of oocytes decreased after IVM.



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  		FIG. 3. The ATP content of nonmatured (A) and in vitro-matured (B) domestic cat oocytes obtained from different animals. Means (bars) and SEMs (error bars) from 15 to 20 oocytes analyzed per experimental group were compared using ANOVA followed by Bonferroni post-hoc test. Within the graph, means marked by different superscripts are significantly (P < 0.05) different

ATP Levels During Embryonic Development In Vitro

Data regarding ATP levels during embryonic development in vitro are presented in Figure 4. The ATP content of two-cell embryos was 1.17 ± 0.08 pmol, which increased to 1.47 ± 0.19 and 1.88 ± 0.32 pmol at the four- and eight-cell stages, respectively. The ATP content then decreased to 1.48 ± 0.10 pmol in 16-cell embryos before reaching a minimum of 0.49 ± 0.04 pmol at the morula stage. Blastocysts, expanded blastocysts (both 144 hpi), and hatching blastocysts (192 hpi) revealed ATP levels of 1.05 ± 0.09, 1.79 ± 0.01, and 4.17 ± 0.21 pmol (P < 0.001 vs. all other stages), respectively.



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  		FIG. 4. The ATP content of different stages of in vitro-produced domestic cat embryos. Between 17 and 31 embryos of each stage from at least three replicates were investigated. Means marked by different superscripts are significantly different (P < 0.05, ANOVA followed by Bonferroni post-hoc test). *P = 0.07 (four-cell stage vs. morula stage)

Effect of ERZP on ATP Content, Total Cell Number, and Developmental Potential

After assisted-hatching treatment (144 hpi) of embryos, ATP content and total cell numbers of blastocysts (4.15 ± 0.37 pmol of ATP, 328.3 ± 48.5 cells) and expanded blastocysts (5.81 ± 0.54 pmol of ATP, 430.1 ± 29.7 cells) analyzed at 192 hpi were significantly (P < 0.001) higher than in their nontreated counterparts (blastocysts: 1.00 ± 0.09 pmol of ATP, 65.3 ± 4.6 cells; expanded blastocysts: 1.79 ± 0.11 pmol of ATP, 121.4 ± 6.5 cells) (Fig. 5). Similarly, at 192 hpi, the ATP content and total cell numbers (4.17 ± 0.21 pmol of ATP, 295.9 ± 20.6 cells) of nontreated hatching blastocysts were significantly (P < 0.05) lower than those of pronase-treated expanded blastocysts.



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  		FIG. 5. Effect of ERZP on total cell number (A) and the ATP content (B) of blastocysts and expanded blastocysts. Embryos were treated (+) at 144 hpi, evaluated at 192 hpi, and compared with nontreated (-) blastocysts and expanded blastocysts evaluated at the same time. Means (bars) and SEMs (error bars) of the ATP content from 23 to 38 embryos analyzed per experimental group (ANOVA followed by Bonferroni post-hoc test) are shown. The means of the total cell numbers (21–95 embryos) were compared using Mann-Whitney U-test. *P < 0.001

Of 195 nontreated blastocysts and expanded blastocysts at 144 hpi, 54 (29.7%) developed to the hatching blastocyst stage at 192 hpi. Of 38 pronase-treated blastocysts and expanded blastocysts at 144 hpi, 37 embryos (97.4%) showed no sign of degeneration, and only one blastocyst (2.6%) collapsed at 192 hpi. Images of assisted-hatching expanded blastocysts before and after staining are presented in Figure 6.



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  		FIG. 6. Representative pictures of a nonstained (A) and a Hoechst 33342-stained (B) blastocyst recovered at 192 hpi after treatment with pronase at 144 hpi. Total cell number = 419, x400

A Spearman-Rho correlation coefficient of 0.61 indicated an overall significant (P < 0.001) correlation between ATP content and total cell number of embryos at 192 hpi. The mean ATP content per cell of expanded blastocysts from the assisted-hatching group showed a tendency (P = 0.27) toward higher values than in nontreated expanded blastocysts (0.13 ± 0.03 fmol vs. 0.10 ± 0.01 fmol) (Fig. 7).



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  		FIG. 7. Effects of assisted-hatching treatment on the ATP content per cell of expanded blastocysts (EBl). Means (bars) and SEMs (error bars) of 24 expanded blastocysts analyzed per experimental group were compared using Student t-test

DISCUSSION

In this study, we investigated, to our knowledge for the first time, the ATP content of nonmatured and in vitro-matured domestic cat oocytes and the ATP content of different stages of in vitro-produced domestic cat embryos. Furthermore, ATP content and total cell numbers of blastocysts and expanded blastocysts that were enzymatically treated to remove the zona pellucida were evaluated.

Production of ATP and accumulation/metabolism of energy are crucial processes for activation and fertilization of oocytes and for embryonic development [3, 4, 6–12, 35–37]. Interestingly, the present study demonstrated that nonmatured domestic cat oocytes contain more ATP than found in oocytes after IVM. This is the opposite of what we found in bovine oocytes [4], suggesting species-specific differences between the follicular environment and its effects on the metabolism of oocytes. Previously, a higher activity of glycolysis as well as of glucose and lactate oxidation was demonstrated for in vitro-matured compared with nonmatured domestic cat oocytes, but glycolysis as well as glucose and palmitate oxidation were increased in the in vivo- versus in vitro-matured domestic cat oocytes [12]. In that study, the authors used 1.64 IU/ml of ovine FSH in the maturation medium. The different maturation conditions used for bovine and domestic cat oocytes could also be responsible for differences in the energy status. Bovine oocytes were matured in the presence of ECS, whereas the maturation medium for domestic cat oocytes was supplemented with BSA. Additionally, the concentration of bovine FSH used for the maturation of domestic cat oocytes (0.02 IU/ml) was twice that used for bovine oocytes. The effects of hormones on the energy metabolism of domestic cat oocytes are still not known. Bovine oocytes matured with LH supplementation showed increased glycolytic activity and oxidation of glucose and glutamine [37]. However, conflicting reports still remain concerning oocyte glucose oxidation in different species [12]. The effects of diet and season on the metabolic activity and energy levels of domestic cat oocytes are not known, but our study clearly demonstrates that the ATP content of nonmatured and matured oocytes is influenced by the donor, as shown in humans [7].

The ATP content of two-cell embryos was lower than that of nonmatured and matured oocytes but increased at the four- and eight-cell stages. The ATP content then decreased between the eight-cell stage and the morula stage, but it increased again at the blastocyst and expanded blastocyst stages and, particularly, at the hatching blastocyst stage. Similar results were obtained after measurements of ATP levels at different stages of in vitro-cultured mouse embryos [38]. This oscillation between steady states is probably caused by the changes of mitochondrial structure and activity, by different metabolic pathways, and by the energy source available during the different stages of maturation and cleavage [9, 26, 34, 38–41]. In mice, it was postulated that a high total ATP content is related to a low-energy state (i.e., low levels of ATP synthesis), and that increased ATP content could be associated with decreased nucleic acid and protein synthesis [38]. In the bovine, the transcriptional activity, as measured by 35S-uridine triphosphate incorporation was high in nonmatured oocytes, decreased sharply in metaphase II oocytes, remained at the same level up to the four-cell stage, but then increased during the eight-cell stage [42]. Regarding in vitro-produced domestic cat embryos, {alpha}-amanitin, an inhibitor of transcription, blocks embryonic development at the five- to eight-cell stage, which is the same stage at which nuclear labeling by 3H-uridine incorporation is first observed [43]. Additionally, the rate of protein degradation in early bovine embryos was higher than that of protein synthesis, with total protein content increasing at compaction of morulae [44]. The fluctuation in ATP levels from the immature oocyte to the hatching blastocyst of the domestic cat, with the lowest level occurring at the morula stage, is similar to previously described ATP levels in ovine oocytes and embryos [45]. The patterns are indicative of different metabolic activity and nutritional requirements of oocytes and embryos of different stages.

In mammalian embryos, a combination of lysins produced by the blastocyst or the uterus and/or physical expansion of the blastocoel reduces the zona thickness in preparation for hatching. The relationship between total ATP content of embryos and the rates of blastocoel formation and hatching is not clear. Fluid accumulation in the blastocoel of rat embryos does not require ATP generated by oxidative phosphorylation, but the blastocysts may increase glucose consumption and metabolism by glycolysis [46]. In contrast, ATP production via oxidative phosphorylation is essential for bovine embryonic development in vitro [47, 48]. Furthermore, IVF of bovine oocytes with a high ATP content [4] and embryo culture in the presence of coenzyme Q10 [35], an important electron carrier, resulted in increased frequencies of development to blastocysts, along with higher ATP levels, higher total cell numbers, and an increased hatching rate. A striking finding of the present study was the marked increase in ATP levels and cell numbers of in vitro-produced blastocysts from which the zona pellucida was enzymatically removed. Whether this has any relevance for improving the embryo survival rate after embryo transfer, however, remains open. Culture conditions may alter the physiological properties of domestic cat embryos, inhibiting spontaneous zona dissolution and, eventually, impairing other processes that are important for successful early embryonic development.

In summary, our study investigated, to our knowledge for the first time, changes in ATP levels of oocytes before and after IVM and in different stages of in vitro-produced domestic cat embryos. The ERZP of blastocysts resulted in a marked increase of ATP content and cell numbers. In addition to the nutritional requirements of the embryo proper, the mechanical and biochemical properties of the zona pellucida in vitro versus in vivo deserve further investigation to optimize systems for in vitro production of cat embryos.

FOOTNOTES

First decision: 7 March 2001.

1 Correspondence: M. Stojkovic, Department of Molecular Animal Breeding and Biotechnology, Hackerstr. 27, 85764 Oberschleissheim, Germany. FAX: 49 89 315 2799; m.stojkovic{at}gen.vetmed.uni-muenchen.de Back

Accepted: April 19, 2001.

Received: February 2, 2001.

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R. M. Rivera, G. M Dahlgren, L. A. de Castro e Paula, R. T Kennedy, and P. J Hansen
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