Theriogenology xxx (2014) 1–7
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Effect of dexamethasone on development of in vitro– produced bovine embryos Priscila P.B. Santana a, *, Carla M.F. Carvalho a, Nathália N. da Costa a, Thiago V.G. Silva a, Priscilla C.A. Ramos a, Marcela S. Cordeiro b, Simone S.D. Santos a, André S. Khayat a, Otávio M. Ohashi a, Moysés S. Miranda a a b
Institute of Biological Sciences, Federal University of Pará, Belém, Para, Brazil Federal Institute of Education, Science and Technology of Pará, Abaetetuba, Para, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 December 2013 Received in revised form 5 February 2014 Accepted 15 February 2014
Studies in somatic cells have shown that glucocorticoids such as dexamethasone (DEX) may trigger or prevent apoptosis depending on the cell type in culture. Because the dysregulation of apoptosis may lower in vitro embryo production efficiency, we sought to investigate the effects of supplementing IVC medium with DEX (0.1 mg/mL) on embryo morphology, development kinetics, and apoptosis rates of in vitro–produced bovine preimplantation embryos. Embryo morphology was graded on Day 7, and development rates were assessed on Days 4 and 7 of IVC. Apoptosis was evaluated via annexin/propidium iodide staining under fluorescence microscopy where a cell labeled with annexin, propidium iodide, or both would be considered apoptotic. An embryo was counted in the apoptosis rates, if it displayed at least one such labeled cell. Although DEX supplementation did not reduce apoptosis rates, it had a positive impact on developmental kinetics and cell number both on Days 4 and 7 of embryo culture. Presumably, such effect resulted from increased cell proliferation rather than a direct inhibition of apoptosis. Further studies may evaluate the mechanisms by which glucocorticoids may affect embryo development, as DEX supplementation could become a tool to improve in vitro embryo yield in mammalian species. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: In vitro embryo production Bovine Dexamethasone Apoptosis
1. Introduction Although the onset of programmed cell death or apoptosis is a physiological event playing a role in cell selection in preimplantation mammalian embryos [1,2], the regulation of this process may be altered in in vitro– production systems. For instance, the percent of apoptotic cells was higher in in vitro than in vivo–derived bovine blastocysts suggesting a negative effect of gamete manipulation and/or culture conditions on embryo health [3,4]. Moreover, in vitro conditions predisposing to embryo * Corresponding author. Tel./fax: þ55 91 32017773. E-mail address: [email protected] (P.P.B. Santana). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.02.017
apoptosis also gave rise to defective mouse embryos that failed to develop normally after transfer [5]. This could be particularly relevant in livestock species when dealing with valuable embryos or those resulting from SCNT. In fact, SCNT-derived embryos may be particularly predisposed to undergoing apoptosis, thus further lowering the efficiency of this technology [6–8] Apoptosis in in vitro– production systems is likely caused, at least partially, by suboptimal culture conditions, resulting in embryos that often display abnormal or delayed cleavage, ultimately affecting the percent and quality of the blastocysts produced [2,9–11]. Hence, media additives inhibiting apoptosis could have an important positive impact in in vitro embryo production rates.
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In this regard, glucocorticoids (GCs) are biological compounds affecting a wide array of metabolic functions exerted both via the genomic regulation of cellular metabolism [12] and cell cycle [13], and through nongenomic effects on cell metabolism and signaling (reviewed by [14]). Dexamethasone (DEX), is a synthetic GC that via its effects on cell-to-cell contact and signaling pathways prevented apoptosis in bovine granulosa cells [15–18] and in human lung epithelial cells [19], mammary epithelial cells [20], and hepatocytes [21]. Conversely, however, DEX also stimulated apoptosis in thymocytes and lymphocytes, an effect that correlated with the expression of an intracellular glucocorticoid receptor [14,22]. Although there is evidence for GCs inhibiting apoptosis through both genomic and nongenomic pathways, the mechanisms are still unclear [23]. For instance, some studies have addressed the correlation between the expression of Bcl-2 antiapoptotic proteins and cell sensitivity to GC-induced apoptosis [21,24,25]; in this regard, DEX differentially regulated Bcl-2 transcription levels in human neutrophils and eosinophils, resulting in a delay and acceleration of apoptosis, respectively [26]. Interestingly, however, DEX activated survival pathways in bovine granulosa cells via Mitogen-activated protein kinase and Protein Kinase B (Akt/PKB) phosphorylation, suggesting a nongenomic-driven effect of GCs mediated through their interaction with a putative membrane bound receptor [15]. Conversely, DEX-mediated MAPK pathway downregulation induced apoptosis in multiple myeloma cells [27]. Altogether, these studies suggest that the dichotomy observed with GCs in regards to the induction or prevention of apoptosis may depend partially on cell type, and on GC receptor or apoptotic gene expression, and/or prosurvival signaling pathway activity in the particular cell population under study, among other factors, given the widespread range of effects known to be exerted by GCs both in vivo and in vitro [23]. Given the above premises, the aim of this study was to investigate the effects of DEX addition during IVC of bovine embryos, and in particular in regards to its ability to modulate apoptosis. 2. Materials and methods 2.1. Reagents Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). 2.2. In vitro embryo production Cumulus-oocyte complexes (COCs) were recovered from small antral follicles (2–8 mm) by follicular aspiration of abattoir ovaries. Compact COCs showing at least two layers of cumulus cells and a homogenous and refringent cytoplasm were selected for IVM [28]. The IVM medium was Tissue culture medium 199 supplemented with 2.2 g/L sodium bicarbonate, 10% Fetal bovine serum (Gibco BRL, Grand Island, NY, USA), 0.5 mg/mL FSH (Folltropin; Bioniche Animal Health, Belleville, Ontario, Canada), and 5.0 mg/mL LH (Lutropin; Bioniche Animal
Health). The COCs were incubated for 18 hours under 5% CO2 in humidified air at 38.5 C. Frozen-thawed bull semen (Bos taurus taurus) was prepared for IVF by Percoll (GE Healthcare Bio-Sciences, Uppsala, Sweden) density gradient centrifugation at 180 g for 7 minutes. For IVF, 2 106 spermatozoa per mL and 20 oocytes were coincubated for 27 hours in 80-mL droplets of a modified Fert-Tyrode’s albumin lactate pyruvate medium [29] supplemented with 30 mg/mL heparin, 1.8 mM epinephrine, 18 mM penicillamine, 10 mM hypotaurine, and 4 mg/mL BSA. After IVF, groups of 20 presumptive zygotes were transferred to 100-mL droplets of modified synthetic oviductal fluid medium as previously described [30]. IVF was followed by 7 days of IVC with no replacement of medium during this period. Both IVF and IVC were performed under 5% CO2 in humidified air at 38.5 C. 2.3. Dexamethasone treatment dose titration Three concentrations of DEX (0.01, 0.1, and 1 mg/mL) were initially evaluated for their effect in IVC of bovine embryos. For this purpose, DEX was diluted in distilled sterile water to make a 10 mM stock solution (following manufacturer’s recommendations) that then was further diluted to obtain the final concentrations tested. Blastocyst rates, total cell number, and embryo quality were end points evaluated on Day 7 of culture. On Day 7 of culture, embryos were assessed for morphologic quality using the criteria established by Stringfellow and Seidel [31]. Thus, grade 1 or very good quality embryos had an inner cell mass (ICM) with symmetrical or few very slightly asymmetrical blastomeres, that displayed a light appearance. Grade 2 or fair quality embryos had an ICM with very slightly asymmetrical blastomeres. And, grade 3 or poor quality embryos had an ICM that displayed widespread marked asymmetry with a darker appearance. Grade 1 and 2 embryos were pooled for analysis herein based on previous evidence that embryos in both categories yield significantly high pregnancy rates, and are preferentially selected for transfer, whereas grade 3 embryos typically yield low pregnancy rates and thus are not preferentially selected for transfer [32]. 2.4. Experiment I: Effect of DEX on embryo development and quality Cleavage and blastocyst rates were evaluated on Days 4 and 7 of IVC, respectively (the fertilization day was set as Day 0). Blastocysts were classified as early, expanded, or hatched [31]. After evaluation, embryos were fixed in 1% formol-saline solution and stained with the DNA dye Hoechst 33342 (5 mg/mL) in PBS. Total cell number was counted using an inverted fluorescence microscope (Eclipse TE 300; Nikon Instruments Inc., Melville, NY, USA). 2.5. Experiment II: Morphology assessment and analysis of apoptosis in early embryos treated with DEX On Day 4, embryos were evaluated for cleavage using phase contrast inverted microscopy and classified as slow (up to 12 blastomeres) or fast cleaving (more than 13
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blastomeres) according to the number of cells. Classification of embryos as slow or fast cleaving was based on data reported by Leidenfrost, et al. [33] regarding the development kinetics of early bovine embryos. Immediately after morphologic assessment, embryos were stained using an annexin V/propidium iodide (PI) assay (Molecular Probes, Inc., Eugene, OR, USA) for apoptosis analysis, following the manufacturer’s instructions. Briefly, embryos were rinsed in 1X binding buffer and incubated with annexin V (Fluorescein isothiocyanate-labeled) for 15 minutes at 4 C. Embryos were incubated with the PI solution for another 15 minutes at 4 C, then stained with Hoechst 33342 (5 mg/mL), and mounted on slides for evaluation under fluorescence microscopy (Olympus BX41; Olympus America Inc., Melville, NY, USA). The embryos were classified as apoptotic or nonapoptotic. Hence, if an embryo had one or more apoptotic blastomeres, it was considered apoptotic according to the criteria proposed by Matwee, et al. [34] and hence counted within our apoptotic rates’ category. Apoptotic blastomeres would be those stained positive for annexin V, PI, or both annexin and PI. Only unstained embryos were included in the nonapoptotic category. 2.6. Statistical analysis Data for cleavage and blastocyst rates and for total cell number were analyzed using ANOVA and Tukey or Student–Newman–Keuls’ post hoc tests when required. Data for morphologic assessment and apoptosis evaluation on Day 4 of IVC were analyzed using the c2 test. Apoptotic rates were based on the number of embryos showing at least one apoptotic cell. All analyses were performed with SigmaPlot 11.0 software (Systat Software, Inc., Erkrath, Germany) [35]. A P-value of 0.05 or less denoted significance. 3. Results 3.1. DEX treatment: A dose-response titration experiment As compared with the untreated control, DEX treatment had no effect (P > 0.05) on bovine embryo cleavage or blastocyst rates (Table 1). However, culture in the presence of 0.1 mg/mL DEX yielded the highest percent (53.9%; P 0.05) of good quality embryos (Table 2). Based on these results, 0.1 mg/mL DEX was chosen for further experiments. Table 1 Development rates of in vitro–produced bovine embryos cultured in the presence of three concentrations of dexamethasone. Group
Oocytes (n)
Development rates (%; mean SEM) Cleavagea
Control 0.01 mg/mL 0.1 mg/mL 1 mg/mL
119 122 121 113
88.1 89.4 83.2 85.4
4.3 2.6 4.0 2.7
Blastocysta 50.8 46.7 49.8 43.0
The results are average of at least five independent experiments. Abbreviation: SEM, standard error of the mean. a Rates based on total oocyte number.
4.1 3.6 2.9 3.2
3
Table 2 Quality grading of bovine blastocysts cultured in the presence of three concentrations of dexamethasone. Group
Embryo quality (%; mean SEM)
Control 0.01 mg/mL 0.1 mg/mL 1 mg/mL
74.9 80.6 46.0 60.5
Grade 3
1.8a,d 1.5a,d 3.1b,d 2.6c,d
Grade 2/1 25.0 19.4 53.9 39.5
1.8a,e 1.5a,e 3.1b,e 2.6c,e
The results are the average of at least five independent experiments. a,b,c Different superscript letters denote significant differences (P 0.05) within column. d,e Different superscript letters denote significant differences (P < 0.05) within row. Abbreviation: SEM, standard error of the mean.
3.2. Experiment I: Effect of DEX on embryo development and quality We next sought to analyze the effect of embryo culture in the presence of 0.1 mg/mL DEX on the kinetics of blastocyst development. Treatment with DEX yielded similar percentages of early, expanded, and hatched blastocysts than those observed in the untreated group (P > 0.05). Notably, embryo culture in the presence of DEX did result in expanded and hatched blastocysts presenting higher cell numbers (P 0.05; Table 3), consistent with a positive effect on development. 3.3. Experiment II: Effect of DEX on the morphology and apoptosis rates of Day 4 (early) embryos To ascertain whether the positive effects of DEX on cell number related to its ability to prevent apoptosis, we next evaluated cleavage kinetics and apoptosis rates in early (Day 4) embryos. Thus, IVC in DEX-containing medium yielded a higher percentage of fast-cleaving embryos as compared with the control group (64.3% vs. 23.8%, respectively; P < 0.05; Fig. 1). We then analyzed apoptosis rates thus comparing the percentages of slow- and fast-cleaving embryos between DEX-treated and untreated groups (Table 4). Interestingly, the percent of apoptotic embryos was exactly the same in both experimental groups (28%). Similarly, no differences in apoptosis rates were detected within morphologic groups (slow- or fast-cleaving embryos). Additionally, the number of apoptotic cells per embryo was similar between treated and untreated groups, for both slow- (1.1–1.5 cells) and fast-cleaving embryos (2 cells; Table 5). Shown in Figure 2 are representative images of early bovine embryos classified as fast-cleaving and apoptotic (Fig. 2A, B, E and F) or not apoptotic (Fig. 2C and D), for each of the experimental groups. 4. Discussion In vitro–produced embryos undergo apoptosis at higher rates than their in vivo–produced counterparts [7,36], thus negatively affecting the success rates in in vitro livestock production systems. Given that the occurrence of apoptosis in in vitro–produced embryos appears to be strongly
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Table 3 Effect of IVC in the presence of 0.1 mg/mL of dexamethasone (DEX) on Day-7 bovine embryo quality. Group
Blastocysts (%; mean SEM) Early
Control DEX
e
47.7 7.3c 58.8 9.8c
Expanded
Total cell number (%; mean SEM) e
29.8 2.9c,d 24.3 6.3d
Hatched
e
21.3 5.8d 16.7 6.7e
f
Total n (%)
Early
Expanded
Hatched
80 (40.7) 69 (34.5)
64.7 4.8c 74.0 2.3a
84.7 2.0a,d 117.6 3.4b,d
128.2 4.1a,e 153.7 9.7b,e
The results are average of at least five independent experiments. a,b Different superscript letters denote significant differences (P < 0.05) within column. c,d Different superscript letters denote significant differences (P < 0.05) within row. Abbreviations: DEX, dexamethasone; SEM, standard error of the mean. e Percentage based on the total number of blastocysts. f Percentage based on the total number of oocytes inseminated.
associated with the culture conditions [3,10,37] and that DEX has been shown to inhibit apoptosis in some somatic cell systems [19–21], we hypothesized that DEX supplementation during IVC would have a positive impact on embryo development. However, although DEX treatment had no quantitative effect on cleavage/blastocyst or apoptosis rates, treatment improved developmental kinetics and increased the number of cells both on Days 4 and 7, suggesting some positive effects of DEX on cell proliferation. In agreement, recent studies on fish embryos have shown similar positive effects of GCs via the expression enhancement of IGF-related growth factor genes [38,39]. Additionally, fasted rats displayed stress-induced plasma increases in GC concentrations that correlated with increases in the expression of the cell cycle regulation proteins p21 and cyclin D1 in gastric and intestinal mucosa, supporting another possible mechanism by which GCs may induce cell proliferation [13]. Interestingly, the GC receptors that are speculated to mediate DEX effects in somatic cell studies are also expressed in bovine embryos from Day 2 of development [40]. In this regard, we speculate that the stressful condition of embryo culture in itself [41] may facilitate the effects of DEX on cell proliferation via both genomic and nongenomic mechanisms. In support of this hypothesis, previous studies have shown that DEX can bind stress-activated GC receptors in mitochondria thereby regulating the membrane potential in hippocampal neurons
[42]. This may also result in GC-driven upregulation of energy metabolism [43,44], which in turn is essential in driving cell proliferation in mammalian embryos [45]. Considering the wide range of effects reported for GCs on somatic cells, future studies may be aimed at analyzing the mechanisms involved in DEX-induced cell proliferation of IVC embryos. Moreover, the above reported findings beg the question of whether stressing IVF-produced zygotes before IVC may allow DEX treatment to further exert a positive effect on development. Given the reported antiapoptotic effects of DEX in somatic cell culture, we hypothesized that its addition to the IVC system would reduce apoptotic rates in in vitro–produced bovine embryos. However, rates of apoptosis were similar between untreated and treated embryos. Worth noting is that the rates of apoptosis shown herein were similar to those previously reported, which, in turn, were highly correlated to the stage of embryo development [7,8,34,46]. In fact, apoptosis as assessed via TUNEL assay was first detected in 30% to 50% of in vitro–produced embryos just after the eight- to 16-cell stage [34]. Herein, using a different apoptotic marker (annexin V), we observed similar rates (w30%) at the same stage of development, thus corroborating the occurrence of apoptosis during early in vitro embryo development in bovine embryos. Conversely, however, apoptosis as detected by TUNEL occurred as early as the four-cell stage in SCNT bovine embryos. Moreover, such embryos displayed higher apoptosis rates throughout all stages of development as compared with IVF embryos [6,8]. Thus, future studies should also test the effects of DEX treatment on SCNT embryo development. One additional consideration is that of how early embryo apoptosis may affect their further developmental potential [33,34]. To some extent, apoptosis may be
Table 4 Effect of addition of 0.1 mg/mL dexamethasone (DEX) to the IVC medium on the development kinetics and apoptosis rates of Day 4 bovine embryos. Group
Slow a
Control DEX Fig. 1. Effect of in vitro culture medium containing 0.1 mg/mL dexamethasone upon the developmental kinetics of early bovine embryos. Slowcleaving embryos (black bar) displayed less than 12 cells. Fast-cleaving embryos (white bar) displayed more than 13 cells. * denotes a significant difference (P 0.05).
Fast
n
Apoptotic
16 5
31 (5/16) 40 (2/5)
b
n 5 9
a
Total Apoptotic 20 (1/5) 22 (2/9)
b
na
Apoptoticb
21 14
28 (6/21) 28 (4/14)
The results are average of at least five independent experiments. No difference was found between the experimental groups (P 0.05; c2 test). Abbreviation: DEX, dexamethasone. a Number of embryos analyzed at each experimental group. b Percentage of embryos containing at least one apoptotic blastomere.
P.P.B. Santana et al. / Theriogenology xxx (2014) 1–7 Table 5 Addition of 0.1 mg/mL dexamethasone to the IVC medium on the total number of cells and number of apoptotic blastomeres of Day 4 bovine embryos. Groupa
Control (mean SEM)
DEX (mean SEM)
Slow
Fast
Slow
Fast
No. of cells Apoptotic
7.4 0.7b,d 1.1 0.1c
13 0.4b,e 2.0 0.5c
6.4 1.3b,d 1.5 0.5c
16.4 1.2b,e 2.0 0.0c
The results are average of at least five independent experiments. b,c Different superscript letters denote significant differences (P 0.05) within column. d,e Different superscript letters denote significant differences (P 0.05) within row. Abbreviations: DEX, dexamethasone; SEM, standard error of the mean. a Number of apoptotic blastomeres in each embryo.
considered a normal mechanism to eliminate abnormal cells which could otherwise impair later embryo development [1], that is, as long as remaining cells would be able to compensate for cells lost early in the cleavage process [2,47]. Therefore, high rates of apoptosis in the early embryo are also related to embryo losses, a fairly frequent occurrence in mammals which is typically manifested as infertility [48,49]. Notably, although apoptosis was detected in early stages of embryo development in our study, no adverse outcome was observed in regards to blastocyst rates on Day 7 of IVC, with or without DEX treatment. This finding is consistent with early apoptosis not impairing later embryo development. Moreover, previous findings
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support the notion of apoptosis being a potentially reversible process, at least up to a certain threshold; however, neither this threshold nor the reversibility mechanism has been elucidated [50,51]. Notably, an SCNT pregnancy was obtained through the use of annexin Vpositive bovine fibroblasts suggesting that the apoptotic stimulus initiated in the donor cell was reversed within the oocyte’s cytoplasm, hence resulting in competent embryos. However, not all annexin V-positive cells yielded embryos, suggesting a heterogeneity in the apoptotic process among cells [52]. In fact, annexin V binding to detect phosphatidylserine externalization is generally considered a very sensitive detection method of irreversible early apoptosis [53]. However, in this study, Day 4 apoptotic embryos treated with DEX developed to Day 7 with higher cell numbers as compared with untreated embryos. Hence, whether apoptosis was reversed in such embryos, or DEX simply compensated for this process by increasing cell proliferation is unclear. To further investigate these assumptions, embryos with or without apoptotic cells should be cultured separately in the presence or absence of DEX and followed through until Day 7 of development. 4.1. Conclusions In summary, our results are consistent with DEX having a positive impact on bovine embryo developmental kinetics through increased cell proliferation rather than
Fig. 2. Representative images of early (Day 4) bovine embryos cultured with or without 0.1 mg/mL dexamethasone (DEX)-containing medium. Treatment increased the percentage of fast-cleaving embryos (C, D, E, F) in comparison to the untreated control group (A, B). Unstained embryos were considered nonapoptotic (C, D). Embryos were considered apoptotic if they had at least one blastomere stained with annexin (B, F; green color indicated by arrow), propidium iodide (image not shown), or with annexin plus propidium iodide (F; orange color indicated by arrowhead). Brightfield (A, C, E), and epifluorescence counterstained with the nuclear dye Hoechst 33342 (B, D, F, corresponding to A, C, E, respectively). 200X magnification.
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Effect of dexamethasone on development of in vitro– produced bovine embryos Priscila P.B. Santana a, *, Carla M.F. Carvalho a, Nathália N. da Costa a, Thiago V.G. Silva a, Priscilla C.A. Ramos a, Marcela S. Cordeiro b, Simone S.D. Santos a, André S. Khayat a, Otávio M. Ohashi a, Moysés S. Miranda a a b
Institute of Biological Sciences, Federal University of Pará, Belém, Para, Brazil Federal Institute of Education, Science and Technology of Pará, Abaetetuba, Para, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 December 2013 Received in revised form 5 February 2014 Accepted 15 February 2014
Studies in somatic cells have shown that glucocorticoids such as dexamethasone (DEX) may trigger or prevent apoptosis depending on the cell type in culture. Because the dysregulation of apoptosis may lower in vitro embryo production efficiency, we sought to investigate the effects of supplementing IVC medium with DEX (0.1 mg/mL) on embryo morphology, development kinetics, and apoptosis rates of in vitro–produced bovine preimplantation embryos. Embryo morphology was graded on Day 7, and development rates were assessed on Days 4 and 7 of IVC. Apoptosis was evaluated via annexin/propidium iodide staining under fluorescence microscopy where a cell labeled with annexin, propidium iodide, or both would be considered apoptotic. An embryo was counted in the apoptosis rates, if it displayed at least one such labeled cell. Although DEX supplementation did not reduce apoptosis rates, it had a positive impact on developmental kinetics and cell number both on Days 4 and 7 of embryo culture. Presumably, such effect resulted from increased cell proliferation rather than a direct inhibition of apoptosis. Further studies may evaluate the mechanisms by which glucocorticoids may affect embryo development, as DEX supplementation could become a tool to improve in vitro embryo yield in mammalian species. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: In vitro embryo production Bovine Dexamethasone Apoptosis
1. Introduction Although the onset of programmed cell death or apoptosis is a physiological event playing a role in cell selection in preimplantation mammalian embryos [1,2], the regulation of this process may be altered in in vitro– production systems. For instance, the percent of apoptotic cells was higher in in vitro than in vivo–derived bovine blastocysts suggesting a negative effect of gamete manipulation and/or culture conditions on embryo health [3,4]. Moreover, in vitro conditions predisposing to embryo * Corresponding author. Tel./fax: þ55 91 32017773. E-mail address: [email protected] (P.P.B. Santana). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.02.017
apoptosis also gave rise to defective mouse embryos that failed to develop normally after transfer [5]. This could be particularly relevant in livestock species when dealing with valuable embryos or those resulting from SCNT. In fact, SCNT-derived embryos may be particularly predisposed to undergoing apoptosis, thus further lowering the efficiency of this technology [6–8] Apoptosis in in vitro– production systems is likely caused, at least partially, by suboptimal culture conditions, resulting in embryos that often display abnormal or delayed cleavage, ultimately affecting the percent and quality of the blastocysts produced [2,9–11]. Hence, media additives inhibiting apoptosis could have an important positive impact in in vitro embryo production rates.
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In this regard, glucocorticoids (GCs) are biological compounds affecting a wide array of metabolic functions exerted both via the genomic regulation of cellular metabolism [12] and cell cycle [13], and through nongenomic effects on cell metabolism and signaling (reviewed by [14]). Dexamethasone (DEX), is a synthetic GC that via its effects on cell-to-cell contact and signaling pathways prevented apoptosis in bovine granulosa cells [15–18] and in human lung epithelial cells [19], mammary epithelial cells [20], and hepatocytes [21]. Conversely, however, DEX also stimulated apoptosis in thymocytes and lymphocytes, an effect that correlated with the expression of an intracellular glucocorticoid receptor [14,22]. Although there is evidence for GCs inhibiting apoptosis through both genomic and nongenomic pathways, the mechanisms are still unclear [23]. For instance, some studies have addressed the correlation between the expression of Bcl-2 antiapoptotic proteins and cell sensitivity to GC-induced apoptosis [21,24,25]; in this regard, DEX differentially regulated Bcl-2 transcription levels in human neutrophils and eosinophils, resulting in a delay and acceleration of apoptosis, respectively [26]. Interestingly, however, DEX activated survival pathways in bovine granulosa cells via Mitogen-activated protein kinase and Protein Kinase B (Akt/PKB) phosphorylation, suggesting a nongenomic-driven effect of GCs mediated through their interaction with a putative membrane bound receptor [15]. Conversely, DEX-mediated MAPK pathway downregulation induced apoptosis in multiple myeloma cells [27]. Altogether, these studies suggest that the dichotomy observed with GCs in regards to the induction or prevention of apoptosis may depend partially on cell type, and on GC receptor or apoptotic gene expression, and/or prosurvival signaling pathway activity in the particular cell population under study, among other factors, given the widespread range of effects known to be exerted by GCs both in vivo and in vitro [23]. Given the above premises, the aim of this study was to investigate the effects of DEX addition during IVC of bovine embryos, and in particular in regards to its ability to modulate apoptosis. 2. Materials and methods 2.1. Reagents Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). 2.2. In vitro embryo production Cumulus-oocyte complexes (COCs) were recovered from small antral follicles (2–8 mm) by follicular aspiration of abattoir ovaries. Compact COCs showing at least two layers of cumulus cells and a homogenous and refringent cytoplasm were selected for IVM [28]. The IVM medium was Tissue culture medium 199 supplemented with 2.2 g/L sodium bicarbonate, 10% Fetal bovine serum (Gibco BRL, Grand Island, NY, USA), 0.5 mg/mL FSH (Folltropin; Bioniche Animal Health, Belleville, Ontario, Canada), and 5.0 mg/mL LH (Lutropin; Bioniche Animal
Health). The COCs were incubated for 18 hours under 5% CO2 in humidified air at 38.5 C. Frozen-thawed bull semen (Bos taurus taurus) was prepared for IVF by Percoll (GE Healthcare Bio-Sciences, Uppsala, Sweden) density gradient centrifugation at 180 g for 7 minutes. For IVF, 2 106 spermatozoa per mL and 20 oocytes were coincubated for 27 hours in 80-mL droplets of a modified Fert-Tyrode’s albumin lactate pyruvate medium [29] supplemented with 30 mg/mL heparin, 1.8 mM epinephrine, 18 mM penicillamine, 10 mM hypotaurine, and 4 mg/mL BSA. After IVF, groups of 20 presumptive zygotes were transferred to 100-mL droplets of modified synthetic oviductal fluid medium as previously described [30]. IVF was followed by 7 days of IVC with no replacement of medium during this period. Both IVF and IVC were performed under 5% CO2 in humidified air at 38.5 C. 2.3. Dexamethasone treatment dose titration Three concentrations of DEX (0.01, 0.1, and 1 mg/mL) were initially evaluated for their effect in IVC of bovine embryos. For this purpose, DEX was diluted in distilled sterile water to make a 10 mM stock solution (following manufacturer’s recommendations) that then was further diluted to obtain the final concentrations tested. Blastocyst rates, total cell number, and embryo quality were end points evaluated on Day 7 of culture. On Day 7 of culture, embryos were assessed for morphologic quality using the criteria established by Stringfellow and Seidel [31]. Thus, grade 1 or very good quality embryos had an inner cell mass (ICM) with symmetrical or few very slightly asymmetrical blastomeres, that displayed a light appearance. Grade 2 or fair quality embryos had an ICM with very slightly asymmetrical blastomeres. And, grade 3 or poor quality embryos had an ICM that displayed widespread marked asymmetry with a darker appearance. Grade 1 and 2 embryos were pooled for analysis herein based on previous evidence that embryos in both categories yield significantly high pregnancy rates, and are preferentially selected for transfer, whereas grade 3 embryos typically yield low pregnancy rates and thus are not preferentially selected for transfer [32]. 2.4. Experiment I: Effect of DEX on embryo development and quality Cleavage and blastocyst rates were evaluated on Days 4 and 7 of IVC, respectively (the fertilization day was set as Day 0). Blastocysts were classified as early, expanded, or hatched [31]. After evaluation, embryos were fixed in 1% formol-saline solution and stained with the DNA dye Hoechst 33342 (5 mg/mL) in PBS. Total cell number was counted using an inverted fluorescence microscope (Eclipse TE 300; Nikon Instruments Inc., Melville, NY, USA). 2.5. Experiment II: Morphology assessment and analysis of apoptosis in early embryos treated with DEX On Day 4, embryos were evaluated for cleavage using phase contrast inverted microscopy and classified as slow (up to 12 blastomeres) or fast cleaving (more than 13
P.P.B. Santana et al. / Theriogenology xxx (2014) 1–7
blastomeres) according to the number of cells. Classification of embryos as slow or fast cleaving was based on data reported by Leidenfrost, et al. [33] regarding the development kinetics of early bovine embryos. Immediately after morphologic assessment, embryos were stained using an annexin V/propidium iodide (PI) assay (Molecular Probes, Inc., Eugene, OR, USA) for apoptosis analysis, following the manufacturer’s instructions. Briefly, embryos were rinsed in 1X binding buffer and incubated with annexin V (Fluorescein isothiocyanate-labeled) for 15 minutes at 4 C. Embryos were incubated with the PI solution for another 15 minutes at 4 C, then stained with Hoechst 33342 (5 mg/mL), and mounted on slides for evaluation under fluorescence microscopy (Olympus BX41; Olympus America Inc., Melville, NY, USA). The embryos were classified as apoptotic or nonapoptotic. Hence, if an embryo had one or more apoptotic blastomeres, it was considered apoptotic according to the criteria proposed by Matwee, et al. [34] and hence counted within our apoptotic rates’ category. Apoptotic blastomeres would be those stained positive for annexin V, PI, or both annexin and PI. Only unstained embryos were included in the nonapoptotic category. 2.6. Statistical analysis Data for cleavage and blastocyst rates and for total cell number were analyzed using ANOVA and Tukey or Student–Newman–Keuls’ post hoc tests when required. Data for morphologic assessment and apoptosis evaluation on Day 4 of IVC were analyzed using the c2 test. Apoptotic rates were based on the number of embryos showing at least one apoptotic cell. All analyses were performed with SigmaPlot 11.0 software (Systat Software, Inc., Erkrath, Germany) [35]. A P-value of 0.05 or less denoted significance. 3. Results 3.1. DEX treatment: A dose-response titration experiment As compared with the untreated control, DEX treatment had no effect (P > 0.05) on bovine embryo cleavage or blastocyst rates (Table 1). However, culture in the presence of 0.1 mg/mL DEX yielded the highest percent (53.9%; P 0.05) of good quality embryos (Table 2). Based on these results, 0.1 mg/mL DEX was chosen for further experiments. Table 1 Development rates of in vitro–produced bovine embryos cultured in the presence of three concentrations of dexamethasone. Group
Oocytes (n)
Development rates (%; mean SEM) Cleavagea
Control 0.01 mg/mL 0.1 mg/mL 1 mg/mL
119 122 121 113
88.1 89.4 83.2 85.4
4.3 2.6 4.0 2.7
Blastocysta 50.8 46.7 49.8 43.0
The results are average of at least five independent experiments. Abbreviation: SEM, standard error of the mean. a Rates based on total oocyte number.
4.1 3.6 2.9 3.2
3
Table 2 Quality grading of bovine blastocysts cultured in the presence of three concentrations of dexamethasone. Group
Embryo quality (%; mean SEM)
Control 0.01 mg/mL 0.1 mg/mL 1 mg/mL
74.9 80.6 46.0 60.5
Grade 3
1.8a,d 1.5a,d 3.1b,d 2.6c,d
Grade 2/1 25.0 19.4 53.9 39.5
1.8a,e 1.5a,e 3.1b,e 2.6c,e
The results are the average of at least five independent experiments. a,b,c Different superscript letters denote significant differences (P 0.05) within column. d,e Different superscript letters denote significant differences (P < 0.05) within row. Abbreviation: SEM, standard error of the mean.
3.2. Experiment I: Effect of DEX on embryo development and quality We next sought to analyze the effect of embryo culture in the presence of 0.1 mg/mL DEX on the kinetics of blastocyst development. Treatment with DEX yielded similar percentages of early, expanded, and hatched blastocysts than those observed in the untreated group (P > 0.05). Notably, embryo culture in the presence of DEX did result in expanded and hatched blastocysts presenting higher cell numbers (P 0.05; Table 3), consistent with a positive effect on development. 3.3. Experiment II: Effect of DEX on the morphology and apoptosis rates of Day 4 (early) embryos To ascertain whether the positive effects of DEX on cell number related to its ability to prevent apoptosis, we next evaluated cleavage kinetics and apoptosis rates in early (Day 4) embryos. Thus, IVC in DEX-containing medium yielded a higher percentage of fast-cleaving embryos as compared with the control group (64.3% vs. 23.8%, respectively; P < 0.05; Fig. 1). We then analyzed apoptosis rates thus comparing the percentages of slow- and fast-cleaving embryos between DEX-treated and untreated groups (Table 4). Interestingly, the percent of apoptotic embryos was exactly the same in both experimental groups (28%). Similarly, no differences in apoptosis rates were detected within morphologic groups (slow- or fast-cleaving embryos). Additionally, the number of apoptotic cells per embryo was similar between treated and untreated groups, for both slow- (1.1–1.5 cells) and fast-cleaving embryos (2 cells; Table 5). Shown in Figure 2 are representative images of early bovine embryos classified as fast-cleaving and apoptotic (Fig. 2A, B, E and F) or not apoptotic (Fig. 2C and D), for each of the experimental groups. 4. Discussion In vitro–produced embryos undergo apoptosis at higher rates than their in vivo–produced counterparts [7,36], thus negatively affecting the success rates in in vitro livestock production systems. Given that the occurrence of apoptosis in in vitro–produced embryos appears to be strongly
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Table 3 Effect of IVC in the presence of 0.1 mg/mL of dexamethasone (DEX) on Day-7 bovine embryo quality. Group
Blastocysts (%; mean SEM) Early
Control DEX
e
47.7 7.3c 58.8 9.8c
Expanded
Total cell number (%; mean SEM) e
29.8 2.9c,d 24.3 6.3d
Hatched
e
21.3 5.8d 16.7 6.7e
f
Total n (%)
Early
Expanded
Hatched
80 (40.7) 69 (34.5)
64.7 4.8c 74.0 2.3a
84.7 2.0a,d 117.6 3.4b,d
128.2 4.1a,e 153.7 9.7b,e
The results are average of at least five independent experiments. a,b Different superscript letters denote significant differences (P < 0.05) within column. c,d Different superscript letters denote significant differences (P < 0.05) within row. Abbreviations: DEX, dexamethasone; SEM, standard error of the mean. e Percentage based on the total number of blastocysts. f Percentage based on the total number of oocytes inseminated.
associated with the culture conditions [3,10,37] and that DEX has been shown to inhibit apoptosis in some somatic cell systems [19–21], we hypothesized that DEX supplementation during IVC would have a positive impact on embryo development. However, although DEX treatment had no quantitative effect on cleavage/blastocyst or apoptosis rates, treatment improved developmental kinetics and increased the number of cells both on Days 4 and 7, suggesting some positive effects of DEX on cell proliferation. In agreement, recent studies on fish embryos have shown similar positive effects of GCs via the expression enhancement of IGF-related growth factor genes [38,39]. Additionally, fasted rats displayed stress-induced plasma increases in GC concentrations that correlated with increases in the expression of the cell cycle regulation proteins p21 and cyclin D1 in gastric and intestinal mucosa, supporting another possible mechanism by which GCs may induce cell proliferation [13]. Interestingly, the GC receptors that are speculated to mediate DEX effects in somatic cell studies are also expressed in bovine embryos from Day 2 of development [40]. In this regard, we speculate that the stressful condition of embryo culture in itself [41] may facilitate the effects of DEX on cell proliferation via both genomic and nongenomic mechanisms. In support of this hypothesis, previous studies have shown that DEX can bind stress-activated GC receptors in mitochondria thereby regulating the membrane potential in hippocampal neurons
[42]. This may also result in GC-driven upregulation of energy metabolism [43,44], which in turn is essential in driving cell proliferation in mammalian embryos [45]. Considering the wide range of effects reported for GCs on somatic cells, future studies may be aimed at analyzing the mechanisms involved in DEX-induced cell proliferation of IVC embryos. Moreover, the above reported findings beg the question of whether stressing IVF-produced zygotes before IVC may allow DEX treatment to further exert a positive effect on development. Given the reported antiapoptotic effects of DEX in somatic cell culture, we hypothesized that its addition to the IVC system would reduce apoptotic rates in in vitro–produced bovine embryos. However, rates of apoptosis were similar between untreated and treated embryos. Worth noting is that the rates of apoptosis shown herein were similar to those previously reported, which, in turn, were highly correlated to the stage of embryo development [7,8,34,46]. In fact, apoptosis as assessed via TUNEL assay was first detected in 30% to 50% of in vitro–produced embryos just after the eight- to 16-cell stage [34]. Herein, using a different apoptotic marker (annexin V), we observed similar rates (w30%) at the same stage of development, thus corroborating the occurrence of apoptosis during early in vitro embryo development in bovine embryos. Conversely, however, apoptosis as detected by TUNEL occurred as early as the four-cell stage in SCNT bovine embryos. Moreover, such embryos displayed higher apoptosis rates throughout all stages of development as compared with IVF embryos [6,8]. Thus, future studies should also test the effects of DEX treatment on SCNT embryo development. One additional consideration is that of how early embryo apoptosis may affect their further developmental potential [33,34]. To some extent, apoptosis may be
Table 4 Effect of addition of 0.1 mg/mL dexamethasone (DEX) to the IVC medium on the development kinetics and apoptosis rates of Day 4 bovine embryos. Group
Slow a
Control DEX Fig. 1. Effect of in vitro culture medium containing 0.1 mg/mL dexamethasone upon the developmental kinetics of early bovine embryos. Slowcleaving embryos (black bar) displayed less than 12 cells. Fast-cleaving embryos (white bar) displayed more than 13 cells. * denotes a significant difference (P 0.05).
Fast
n
Apoptotic
16 5
31 (5/16) 40 (2/5)
b
n 5 9
a
Total Apoptotic 20 (1/5) 22 (2/9)
b
na
Apoptoticb
21 14
28 (6/21) 28 (4/14)
The results are average of at least five independent experiments. No difference was found between the experimental groups (P 0.05; c2 test). Abbreviation: DEX, dexamethasone. a Number of embryos analyzed at each experimental group. b Percentage of embryos containing at least one apoptotic blastomere.
P.P.B. Santana et al. / Theriogenology xxx (2014) 1–7 Table 5 Addition of 0.1 mg/mL dexamethasone to the IVC medium on the total number of cells and number of apoptotic blastomeres of Day 4 bovine embryos. Groupa
Control (mean SEM)
DEX (mean SEM)
Slow
Fast
Slow
Fast
No. of cells Apoptotic
7.4 0.7b,d 1.1 0.1c
13 0.4b,e 2.0 0.5c
6.4 1.3b,d 1.5 0.5c
16.4 1.2b,e 2.0 0.0c
The results are average of at least five independent experiments. b,c Different superscript letters denote significant differences (P 0.05) within column. d,e Different superscript letters denote significant differences (P 0.05) within row. Abbreviations: DEX, dexamethasone; SEM, standard error of the mean. a Number of apoptotic blastomeres in each embryo.
considered a normal mechanism to eliminate abnormal cells which could otherwise impair later embryo development [1], that is, as long as remaining cells would be able to compensate for cells lost early in the cleavage process [2,47]. Therefore, high rates of apoptosis in the early embryo are also related to embryo losses, a fairly frequent occurrence in mammals which is typically manifested as infertility [48,49]. Notably, although apoptosis was detected in early stages of embryo development in our study, no adverse outcome was observed in regards to blastocyst rates on Day 7 of IVC, with or without DEX treatment. This finding is consistent with early apoptosis not impairing later embryo development. Moreover, previous findings
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support the notion of apoptosis being a potentially reversible process, at least up to a certain threshold; however, neither this threshold nor the reversibility mechanism has been elucidated [50,51]. Notably, an SCNT pregnancy was obtained through the use of annexin Vpositive bovine fibroblasts suggesting that the apoptotic stimulus initiated in the donor cell was reversed within the oocyte’s cytoplasm, hence resulting in competent embryos. However, not all annexin V-positive cells yielded embryos, suggesting a heterogeneity in the apoptotic process among cells [52]. In fact, annexin V binding to detect phosphatidylserine externalization is generally considered a very sensitive detection method of irreversible early apoptosis [53]. However, in this study, Day 4 apoptotic embryos treated with DEX developed to Day 7 with higher cell numbers as compared with untreated embryos. Hence, whether apoptosis was reversed in such embryos, or DEX simply compensated for this process by increasing cell proliferation is unclear. To further investigate these assumptions, embryos with or without apoptotic cells should be cultured separately in the presence or absence of DEX and followed through until Day 7 of development. 4.1. Conclusions In summary, our results are consistent with DEX having a positive impact on bovine embryo developmental kinetics through increased cell proliferation rather than
Fig. 2. Representative images of early (Day 4) bovine embryos cultured with or without 0.1 mg/mL dexamethasone (DEX)-containing medium. Treatment increased the percentage of fast-cleaving embryos (C, D, E, F) in comparison to the untreated control group (A, B). Unstained embryos were considered nonapoptotic (C, D). Embryos were considered apoptotic if they had at least one blastomere stained with annexin (B, F; green color indicated by arrow), propidium iodide (image not shown), or with annexin plus propidium iodide (F; orange color indicated by arrowhead). Brightfield (A, C, E), and epifluorescence counterstained with the nuclear dye Hoechst 33342 (B, D, F, corresponding to A, C, E, respectively). 200X magnification.
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