Reprod Dom Anim doi: 10.1111/rda.12325 ISSN 0936–6768
The Influence of Morphology, Follicle Size and Bcl-2 and Bax Transcripts on the Developmental Competence of Bovine Oocytes THC De Bem1,2, PR Adona3,4, FF Bressan1, LG Mesquita1, MR Chiaratti5, FV Meirelles1 and CLV Leal1 1
Departamento de Ci^ encias, Faculdade de Zootecnia e Engenharia de Alimentos B asicas, Universidade de S~ ao Paulo, Pirassununga, Brazil; Departamento de Gen etica, Faculdade de Medicina de Ribeir~ ao Preto, Universidade de S~ ao Paulo, Ribeir~ ao Preto, Brazil; 3Universidade Norte do aria Laffranchi, Tamarana, Brazil; 5Departamento de Gen etica e Evolucßa~o, Universidade Federal de S~ ao Paran a, Londrina, Brazil; 4Agropecu Carlos, S~ ao Carlos, Brazil 2
Contents This study analysed two non-invasive oocyte selection methods in relation to in vitro embryo development capacity and expression of apoptosis-related genes. Selection was based on morphological quality of oocytes or follicle diameter. Oocytes were classified as grade I (GI ≥3 layers compact cumulus cells and homogeneous cytoplasm; grade II (GII ≤3 layers compact cells and homogeneous cytoplasm;, and grade III (GIII ≥3 layers, but cells with slight expansion and slightly granulated cytoplasm). Blastocyst development was lower for GII (28.5%) than for GIII (47.7%, p < 0.05), and GI was similar to both (36.9%, p > 0.05). Relative expression of Bcl-2 gene was lower in the GI (1.0, p < 0.05) than in the GII (1.8) and GIII (2.2), which were not different (p > 0.05). There was no difference (p > 0.05) between GI (1.0), GII (0.92) and GIII (0.93) regarding the Bax transcript. However, the Bax and Bcl-2 transcript ratios in GII (Bax; 0.92 and Bcl-2; 1.8) and GIII (Bax; 0.93 and Bcl-2; 2.2) were different (p < 0.05). Regarding oocytes from follicles of different sizes, cleavage and blastocyst rates for 1–3 mm (82.5; 23.7%) were lower (p < 0.05) than for 6–9 mm (95.6; 41.1%), but similar (p > 0.05) to 3–6 mm (93.7; 35.4%), which were not different (p > 0.05). Regarding Bax and Bcl-2 expression, the oocytes were similar (p > 0.05) for 1–3 mm (Bax; 1.0 and Bcl-2; 1.0), 3–6 mm (Bax; 1.0 and Bcl-2; 0.93) and 6–9 mm (Bax; 0.92 and Bcl-2; 0.91). In conclusion, oocyte selection based on morphological appearance does not guarantee the success of embryonic development. Additionally, the absence of apoptosis is not necessarily a benefit for the development of oocytes. Bovine COCs with initial signs of atresia may be used for the in vitro production of embryos, and COCs taken from follicles >3 mm in diameter are better suited to in vitro embryo development.
Introduction The in vitro production of embryos is an important biotechnological technique in the field of animal reproduction, widely used in research, but also in commercial settings. Notwithstanding its widespread use, the in vitro production of embryos associated with follicular aspiration guided by ultrasound (ovum pickup) (Pontes et al. 2012) or ovaries taken from abattoirs, an abundant source of oocytes (Dey et al. 2012), still suffers certain limitations due to the relatively low production of embryos (Rizos et al. 2008; Adona et al. 2011). Several analyses have focused on the interaction between developmental competence and different oocyte characteristics. These include studies focusing on the morphology of cumulus–oocyte complexes (Wasielak and Bogacki 2007; Alvarez et al. 2009; Melka et al. 2010),
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the diameter of the oocyte or the size of the follicle from which they are aspirated (Kauffold et al. 2005; Han et al. 2006), the follicular wave phase (Blondin and Sirard 1995; Mossa et al. 2012; Pancarci et al. 2012), the communication of the oocyte with the cumulus cells (Krisher 2004), the accumulated amount of mRNA among other molecules which have been shown to influence, although to different degrees, the growth and potential of the oocyte to develop to the blastocyst stage (Meirelles et al. 2004; Adona et al. 2011; Boumela et al. 2011; SuttonMcDowall et al. 2012). The selection of oocytes based on morphology was first studied in cattle by Leibfried and First (1979); since then, the cumulus–oocyte complexes used in the in vitro production of embryos of several species are usually selected according to the morphological characteristics of the cells and the appearance of the ooplasm under a stereomicroscope (Leon et al. 2013). However, even when using cumulus–oocyte complexes (COCs) regarded as of high quality using this methodology, such COCs do not necessarily possess the best development capacity (Blondin and Sirard 1995; Li et al. 2009). Together with the morphological selection of cumulus–oocyte complexes, the diameter of the follicle is one of the most commonly used parameters in the prediction of bovine oocyte competence (Anguita et al. 2007). The relationship between the diameter of the follicle and the developmental competence of the oocyte has already been shown (Pavlok et al. 1992), whereby some studies indicate a positive relationship between the diameter of the follicle and the in vitro blastocyst developmental competence (Blondin et al. 1997; Kauffold et al. 2005; Han et al. 2006). Oocytes from follicles smaller than 3 mm in diameter are less competent for the in vitro production embryos when compared with those from larger follicles (Lonergan et al. 1994; Kauffold et al. 2005). However, the diameter does not necessarily indicate whether the follicle is healthy or is already in a state of atresia. The selection of oocytes based on morphology and follicle diameter is a common practice in the in vitro production of embryos, but the effectiveness in determining post-fertilization embryo competence is still uncertain (Ebner et al. 2003). In turn, it is known that the presence of programmed cell death may lead to DNA fragmentation and oocyte degeneration (Takase et al. 1995; Matwee et al. 2000), meaning a significant decrease in fertility of oocytes used for the in vitro production of embryos (Fujino et al. 1996).
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THC De Bem, PR Adona, FF Bressan, LG Mesquita, MR Chiaratti, FV Meirelles and CLV Leal
The Bcl-2 gene family, which includes the Bax (proapoptotic) and Bcl-2 (anti-apoptotic) genes, is involved in the regulation of apoptosis (Yang and Rajamahendran 2002). These two genes in particular play a key role in the occurrence of apoptosis in female germ cells (Kim and Tilly 2004) and are used in the analysis of apoptosis in oocytes and embryos (Yang and Rajamahendran 2002; Pocar et al. 2005; Opiela et al. 2008). Research assessing the developmental competence of oocytes selected according to morphology is quite inconsistent and even contradictory (Yuan et al. 2005; Li et al. 2009). Some demonstrate that COCs with early signs of atresia (e.g. a slight increase in the size of the cumulus and a slight cytoplasmic granulation) show greater developmental potential than those regarded as morphologically healthy (Blondin and Sirard 1995; Alvarez et al. 2009; Li et al. 2009). Most studies on in vitro production of embryos is performed only with grade I oocytes obtained from follicles measuring from 3 to 8 mm in diameter, but we believe that a considerable number of oocytes, which do not strictly fall within this classification standard (Grade I), could still be used in the IVP system. With the aim of maximizing the use of oocytes for the in vitro production of embryos, the purpose of this study was to analyse two non-invasive (morphological quality and follicle size) methods and one invasive (genes related to apoptosis – Bcl-2 and Bax) method in the selection of oocytes, associating them with developmental competency and the presence of DNA fragmentation in the embryos to better understand the role of the origin and oocyte quality on development in vitro and quality of blastocysts.
Material and Methods Chemicals were acquired from the Sigma Chemical Company, St Louis, MO, USA, unless specified otherwise. Selection of cumulus–oocyte complexes (COCs) and follicle classification Bovine ovaries were collected from a commercial abattoir and transported to the laboratory in saline (NaCl 0.9%) with antibiotics (50 lg/ml gentamicin) at 25– 30°C. The ovaries were aspirated (Adona et al. 2008a,b) and the COCs separated according to morphological classes: grade I (GI) with more than three layers of compact cells and a homogeneous cytoplasm; grade II (GII) with up to three layers of compact cells and a homogeneous cytoplasm; and grade III (GIII) with more than three layers of cells with a slight expansion and a slightly granulated cytoplasm (de Loos et al. 1992). COCs were also collected according to follicle diameter. For this purpose, the follicles located in the ovarian cortical region were measured with a calliper and classified in different sizes (1–3, 3–6 and 6–9 mm in diameter). From the different follicles sizes, only grade I COCs were used.
In vitro maturation The COCs selected for the experiments were transferred to the maturation medium [199 medium supplemented with 10% foetal bovine serum, 5.0 lg/ml luteinizing hormone (Lutropin-v, Vetrepharm), 0.5 lg/ml folliclestimulating hormone (Folltropin-v, Vetrepharm), 0.2 mM pyruvate and 50 lg/ml gentamicin]. In vitro maturation was performed for 22 h in 100 ll droplets ( 20 COCs per drop) under mineral oil at 38.5°C and an atmosphere of 5% CO2 in air. In vitro fertilization and culture COCs matured in vitro were fertilized with the thawed semen (Genetica Avancßada, S~ao Carlos, Brazil) from the same bull and batch and prepared according to the Percoll gradient technique (Amersham Pharmacia Biotec). Fertilization (2 9 106 spermatozoa/ml) was conducted in TALP medium (Parrish et al. 1988), supplemented with 2 lM penicillamine, 1 lM hypotaurine, 250 lM epinephrine and 20 lg/ml heparin. COCs and spermatozoa were incubated in 100 ll droplets under mineral oil for 18 h. After this period, the COCs were partially denuded and transferred to an in vitro synthetic oviductal fluid culture medium (Holm et al. 1999) supplemented with 2.5% foetal calf serum, 0.2 mM pyruvate and 50 lg/ml gentamicin. Cleavage rate was analysed after 48 h of culture (D2), blastocyst rate on the seventh day (D7) and blastocyst hatching rate on the ninth day (D9). In vitro culture of embryos was performed at 38.5°C and an atmosphere of 5% CO2 in air. Detection of nuclear fragmentation using the TUNEL technique Hatched embryos (D9) were placed in 3% paraformaldehyde and permeabilized in a PBS solution supplemented with polyvinyl alcohol (PVA), 0.5% Triton X100 (USB) and 0.1% sodium citrate for 1 h at room temperature. The TUNEL technique was conducted using the ‘In Situ Cell Death Detection Kit Fluorescein’ (Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. The embryos were separated in groups for negative control analysis (absence of stained cells), positive control analysis (stained cells) and experimental samples. The analyses were performed under an epifluorescent microscope; the cells stained in green (FITC) were considered as positive TUNEL cells, that is, with fragmented DNA, and a blue stain (Hoechst 33342) indicated the location of the nucleus of the cells. Extraction of RNA and reverse transcription (cDNA) After being selected and denuded, the oocytes were stored at 80°C in calcium- and magnesium-free PBS with 0.1% PVA and 100 Ul/ml RNAse inhibitor (Life Technologies, Grand Island, NY, USA). RNA was extracted from pools of 25 oocytes from each group using an RNeasy Protect Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcription (cDNA) of RNA was performed
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Non-invasive Methods to Enhance in vitro Embryo Production
using an oligo (dT) 12–18 primer according to the manufacturer’s recommendations (Improm-II; Promega, Madison, WI, USA). Samples (20 ll) were then frozen for real-time PCR analysis. Quantitative PCR Relative real-time PCR quantification and amplification of cDNA was performed using the kit Power SYBR Green PCR Master Mix according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA) in triplicate. The thermal cycling conditions included initial sample incubation at 50°C for 2 min and at 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and at 60°C for 45 s using an ABI 7500 instrument (Applied Biosystems). Standard curves were generated for each gene (Bax, Bcl-2 and GAPDH – Table 1) by serial dilution using fivefold dilutions (1:3) to create a 5-point standard curve, and the slope was between 3.3 and 3.6, with an R2 > 0.95. Standard curves were performed in a 20 ll reaction volume [10 ll SYBR Green PCR master mix, 1 ll of each primer (10 lM), 7 ll of nuclease-free water and 2 ll cDNA]. In each sample, the median value of PCR triplicates was considered, and Bax and Bcl-2 data were normalized according to the relative concentration of the expression normalizer, the endogenous 3-phosphate dehydrogenase gene (GAPDH). The calculations of relative quantification were conducted using the comparative cycle threshold (Ct) method, and the PCR product was analysed using a dissociation curve.
Experimental Design Experiment 1: the influence of COCs morphology on the in vitro production of embryos The COCs aspirated from 2 to 8 mm follicles were classified under a stereomicroscope and divided into the three morphological (I, II and III). Subsequent to classification, they were matured (22 h), fertilized (18 h) and cultured in vitro for 9 days (D9 = 216 h post-fertilization) for the analysis of blastocyst, hatching and apoptosis rates. Four replicates were performed for analyses of embryo development. Three replicates were performed for the analysis of DNA fragmentation, and the blastocysts (D9) were analysed under an epifluorescent microscope for the quantification of apoptotic cells (TUNEL+). Experiment 2: the influence of follicle size on the in vitro production of embryos The COCs aspirated from follicles of different diameters (1–3, 3–6 and 6–9 mm) were selected under a
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stereomicroscope, and all the viable oocytes (not degenerated or denuded) were matured (22 h), fertilized (18 h) and cultured in vitro for up to 9 days (D9) for the analysis of the blastocyst development, hatching and apoptosis rates. Four replicates were performed for analyses of embryo development. Three replicates were performed for the analysis of DNA fragmentation, and the blastocysts (D9) were analysed under an epifluorescent microscope for the quantification of apoptotic cells (TUNEL+). Experiment 3: relative expression of Bax and Bcl-2 genes in bovine oocytes from COCs of different morphological qualities and from different follicle diameters The immature COCs were classified according to morphology (GI, GII and GIII) and the diameter of the follicle (1–3, 3–6 and 6–9 mm in diameter). After being classified, they were denuded, split into pools of 25 oocytes for each group in four replicate batches and stored at 80°C. The analysis of the transcripts involved the extraction of the RNA, reverse transcription (cDNA) and the relative quantification of the genes in real-time PCR (Fig. 1). Statistical analysis The variables for cleavage, blastocysts and hatching were transformed according to the arcsin function. Nontransformed numbers were used for the analysis of total cell numbers and TUNEL+ cells. The BIOESTATS 5.0 (Instituto de Desenvolvimento Sustentavel Mamirau a, Tefe, AM, Brazil) program was used for all the analyses, and ANOVA was applied at 5% significance to reveal the differences between the treatments. The Tukey post hoc test was used for significant results in the analysis of variances as a multiple comparison procedure between the groups. The relative quantification of the real-time PCR transcripts was analysed using the 2005 – Beta V19.9 REST (Relative Expression Software Tool; Pfaffl et al. 2002) at a significance level of 5% to reveal the differences between transcripts (www.gene-quantifi cation-info).
Results Experiment 1 COCs from different morphological categories were subjected to fertilization for the analysis of in vitro embryo development and the percentage of TUNEL+ cells (Tables 2 and 3). The cleavage rate was approximately 82% and was similar (p > 0.05) for all COCs
Table 1. Sequences of the gene primers analysed in the different treatments Genes
Primers
Bp
Gene bank
GAPDH
F:50 -CGACTTCAACAGCGACACTCA-30 R:50 -AGCCAAATTCATTGTCGTACCA-30 F:50 -TTTGCTTCAGGGTTTCATCCA-30 R:50 -CCGATGCGCTTCAGACACT-30 F:50 -GAGTCGGATCGCAACTTGGA-30 R:50 -CTCTCGGCTGCTGCATTGT-30
107
NM_001034034.2
126
NM_173894.1
120
NM_001077486.2
BAX BCL2
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THC De Bem, PR Adona, FF Bressan, LG Mesquita, MR Chiaratti, FV Meirelles and CLV Leal
Table 2. Embryo development and blastocyst hatching rates according to morphological classification of COCs
Group GI GII GIII
Oocytes N
Cleavage D2 n (% SD)
Blastocysts D7 n (% SD)
Hached D9 n (% SD)
92 91 88
78 (85 4.0) 73 (80 8.8) 72 (82 4.2)
34 (36.9 1.1)ab 26 (28.5 4.8)b 42 (47.7 5.5)a
31 (91.2 5.2) 22 (84.6 8.3) 37 (88.1 8.4)
SD, standard deviation of the mean. Results of four replicates. Different letters within the same column indicate a difference between treatments.
Table 3. Total cell numbers and the percentage of positive TUNEL cells in the embryos from COCs of different morphological categories
Group
Hached D9 N
Cell numbers n ( SD)
TUNEL+ cells% ( SD)
16 16 18
177 ( 29.6) 205 ( 25.9) 185 ( 33.6)
0.14 ( 0.02) 0.32 ( 0.28) 0.84 ( 0.68)
GI GII GIII
SD, standard deviation of the mean. Results of four replicates.
Table 4. Analysis of the COCs taken from follicles of different sizes in relation to embryo development and blastocyst hatching
Group 1–3 mm 3–6 mm 6–9 mm
Oocytes N
Cleavage D2 n (% SD)
Blastocysts D7 n (% SD)
Hached D9 n (% SD)
80 79 68
66 (82.5 3.5)b 74 (93.7 3.0)ab 65 (95.6 9.5)a
19 (23.7 4.7)b 28 (35.4 6.3)ab 28 (41.2 2.8)a
15 (78.9 11.0) 22 (78.6 11.6) 21 (75.0 10.7)
SD, standard deviation of the mean. Results of four replicates. Different letters within the same column indicate a difference between treatments.
groups analysed. The only difference (p < 0.05) detected in relation to in vitro embryo development (D7 blastocysts) was between the GII (28.5%) and GIII (47.7%) groups. The GI group (36.9%) did not differ from the others (p > 0.05). There were also no differences (p > 0.05) in the percentage of hatched blastocysts (D9) for GI (91.2%), GII (84.6%) and GIII (88.1%) COCs; in the average total cell numbers (177, 205 and 185 for GI, GII and GIII, respectively); and in the percentage of positive TUNEL cells (0.14, 0.32 and 0.84%, for GI, GII and GIII, respectively) in hatched embryos.
Bax
(a)
Bcl-2
(b)
2.0
1.0
0.0
a a
a b
a b
GI
GII
GIII
Cumulus oocyte complex morphogy
Relative expression
Relative expression
3.0
Bax
3.0
Table 5. Analysis of the number of cells and the percentage of positive TUNEL cells in the embryos of COCs from different-sized follicles
Group
Hached D9 N
Cell numbers n ( SD)
TUNEL+ cells % ( SD)
15 16 16
268 ( 22.2) 248 ( 17.7) 237 ( 12.1)
0.36 ( 0.18) 0.30 ( 0.17) 0.23 ( 0.27)
1–3 mm 3–6 mm 6–9 mm
SD, standard deviation of the mean. Results of four replicates.
Experiment 2 The COCs were aspirated from follicles of different diameters for the analysis of in vitro embryo development and the percentage of TUNEL+ cells (Tables 4 and 5). The cleavage and blastocyst rates on day seven in the 1–3 mm group (82.5; 23.7%) were lower (p < 0.05) in relation to the 6–9 mm group (95.6; 41.1%), but did not differ (p > 0.05) from 3 to 6 mm (93.7; 35.4%), and these groups did not differ between themselves (p > 0.05). There was also no difference (p > 0.05) in the hatching rate of the COCs from the 1 to 3 mm (78.9%), 3 to 6 mm (78.6%) and 6 to 9 mm (75.0%) groups. The average number of cells (237 to 268) in the embryos and the percentage of apoptotic cells (TUNEL+) for the 1–3 mm (0.36%), 3–6 mm (0.30%) and 6–9 mm (0.23%) groups did not differ (p > 0.05). Experiment 3 Oocytes with different morphological qualities taken from follicles of different diameters were evaluated for relative quantification of Bax and Bcl-2 transcripts. There was a difference (p < 0.05) in the relative expression of the Bcl-2 gene between the GI (1.0) group and the GII (1.8) and GIII (2.2) groups, but no such difference between the GII and GIII groups (p > 0.05) (Fig. 1a). There was also no difference (p > 0.05) between the GI (1.0), GII (0.92) and GIII (0.93) groups for relative expression of the Bax transcript (Fig. 1a). There was difference (p < 0.05) between the two Bax and Bcl-2 transcripts only in the GII (Bax = 0.92 and Bcl-2 = 1.8) and the GIII groups (Bax = 0.93 and Bcl-2 = 2.2) (Fig. 1a). For oocytes from follicles of different sizes (Fig. 1b), the relative expression of Bax and Bcl-2 genes did not vary (p > 0.05) between groups 1–3 mm (Bax = 1.0 and Bcl-2 = 1.0), 3–6 mm (Bax = 1.0 and
Bcl-2
2.0
1.0
0.0 1–3
3–6
6–9
Oocytes from follicles of different sizes
Fig. 1. Relative expression of Bax and Bcl-2 genes in oocytes separated by morphological criteria (a) and oocytes taken from different-sized follicles (b)
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Non-invasive Methods to Enhance in vitro Embryo Production
Bcl-2 = 0.93) and 6–9 mm (Bax = 0.92 and Bcl2 = 0.91), or even between the two (Bax and Bcl-2) genes.
Discussion In the majority of studies, bovine oocytes are considered to be of good quality when they have various compact layers of cumulus cells and homogenous cytoplasm (Wasielak and Bogacki 2007), as the physiological state of the cumulus cells indicated by morphological and physical differences increases the rate of post-fertilization maturation and development (Blondin and Sirard 1995; de Wit and Kruip 2001; Boni et al. 2002). The maturation and posterior development of the oocyte are partially dependent on cumulus cells, which are highly specialized cells and have a transzonal cytoplasmic structures that penetrates the zona pellucida and touches the plasma membrane of the oocyte (Albertini et al. 2001), creating the cumulus oocyte complex (COC). Previous studies examining the embryonic development capacity of oocytes, based on visual appearance, reported that COCs with early signs of atresia (partial expansion of cumulus and small granules in cytoplasm) possess greater embryonic growth potential than COCs that were considered morphologically healthy and of higher quality (de Wit and Kruip 2001; BilodeauGoeseels and Panich 2002; Li et al. 2009). In the present study, the separation of COCs by morphological criteria into GI, GII and GIII groups showed that embryonic development may be affected by the morphology of the oocytes, as the GIII group, considered to be of poor morphological quality, was superior to the intermediate group (GII), but similar to the GI group. However, when the number of cells in the hatched blastocysts and the percentage of positive TUNEL cells were evaluated, no differences were observed between the groups (GI, GII and GIII). Although there was no reduction in the cleavage rate, the percentage of embryos that reached the blastocyst stage was therefore affected by the morphological classification of the COCs, which is in accordance with previous findings (Boni et al. 2002; Li et al. 2009). These results confirm that morphological classification can affect the proportion of blastocysts formed, but not their quality, as COCs classified as GI, GII and GIII did not reveal differences in the number of blastocyst cells and the percentage of positive TUNEL cells. Melka et al. (2010) found few TUNEL+ cells in good quality embryos. This suggests that in this study the embryos from oocytes from GI, GII and GIII groups are of good quality, due to the total number of cells and the low percentage of TUNEL+ cells, and also that the grade of the oocyte does not interfere with such criteria. From this assumption, it may be suggested that the percentage of TUNEL+ cells (6 mm), and these were similar to those from follicles of intermediate diameter (3–6 mm). These results are similar to those reported (Lonergan et al. 1994; Gandolfi and Gandolfi 2001; Kauffold et al. 2005) which state that follicles with diameter >3 mm contain COCs with higher development potential than smaller follicles and the quality of those embryos is closer to those from an in vitro culture system (Rizos et al. 2002; Cui et al. 2011; Sananmuang et al. 2011) as previously mentioned in the present study. The removal of COCs from follicles smaller than 3 mm in diameter before oocyte capacitation is finalized (Hyttel et al. 1996) is, probably, one of the factors involved in the low production of embryos in vitro. Therefore, the interruption of folliculogenesis is responsible for the reduction in oocyte competence. In bovine and ovine, COCs from follicles smaller than 3 mm in diameter have oocytes with a smaller diameter, while follicles >3 mm in diameter have oocytes with a larger diameter (Mirshamsi et al. 2013). The diameter of the follicles affects the expansion of the cumulus cells, in vitro maturation and in the embryonic development of COCs in cattle and sheep (Kauffold et al. 2005; Han et al. 2006). It was also found in sheep that COCs from follicles with diameters smaller than 3 mm had brilliant cresyl blue staining (BCB) inferior to COCs from larger follicles (Mirshamsi et al. 2013), indicating that these COCs are less developmentally competent. Brilliant Cresyl blue staining has been used to evaluate the quality of COCs and their developmental competence (Janowski et al. 2012; Su et al. 2012). In the evaluation of Bcl-2 and Bax transcripts from follicles with different diameters, the expression of these genes and the ratio between them were not affected. Evans et al. (2004) analysed apoptotic genes in granulosa and theca cells during the follicular waves in cows and did not find differences in the ratio of expression of Bcl-2 and Bax between the dominant and subordinate follicles. In another study, the Bcl-2 transcript abundance in GV (germinal vesicle) oocytes was not significantly affected by diameter of the follicle from which the oocytes were derived, but after the IVM culture, the Bcl-2 transcript abundance in MII (metaphase-II) oocytes was significantly affected (Kohata et al. 2013). The transcription abundance of Bcl-2 gene is gradually reduced during ageing of oocytes, resulting in decreasing anti-apoptotic protein Bcl-2 and leading to abnormal sister chromatid segregation during meiosis II, embryo fragmentation and apoptosis (Kohata et al. 2013). Therefore, we believe that the differential expression of the apoptotic genes Bcl-2 and Bax is related only in oocytes with advanced signs of atresia. In this way,
the follicles used during these experiments were likely collected before or immediately after the selection of the dominant follicle and prior to both DNA fragmentation and global activation of the genes involved in the terminal stages of apoptosis (Austin et al. 2001; Evans et al. 2004). The expression of Bax may be constitutive, which suggests that the oocytes are under constant threat and that their development potential depends on their capacity to inhibit pro-apoptotic activity (Boumela et al. 2011). This suggests that changes in gene expression itself are not sufficient to prevent apoptosis. The balance between Bcl-2/Bax in oocytes from differentsized follicles suggests that they have undergone similar development. However, there was a reduction in the level of embryos in the group of COCs from small follicles, but this reduction in the production of embryos may be related to other factors, as previously mentioned in the present study. In conclusion, oocyte selection based on morphological appearance does not guarantee the success of embryonic development. Bovine COCs with initial signs of atresia may be used for the in vitro production of embryos, and COCs from follicles >3 mm in diameter are more suitable for in vitro embryo development than oocytes from smaller follicles. Additionally, the absence of apoptosis is not necessarily a benefit for the development of oocytes; therefore, a moderate apoptosis of the blastocyst may not be harmful for pre-implantational development. The evaluation of Bcl-2 and Bax transcripts is not determinative as biomarkers of the quality of the oocyte and its pre-implantation developmental potential. This suggests that the developmental potential of an oocyte is dependent on numerous variables, and multiple factors must be considered to determine the quality of an oocyte. However, it would be interesting to analyse the blastocysts transfers to observe the relationship between oocyte quality, follicle size and the apoptosis levels with the pregnancy rates and the success of development to term. Acknowledgements The present study was supported by FAPESP, Brazil (Grant # 05/ 59694-5). THCDB and PRA were supported by FAPESP, Brazil (06/ 58536-0 and 07/51953-9, respectively).
Conflict of interest None of the authors have any conflict of interest to declare.
Author contributions THC De Bem contributed with all the in vitro development experiments, carrying out the experiments, data analysis and drafting the paper; PR Adona contributed with the experimental design, carrying out the experiments and drafting the article; FF Bressan contributed with in vitro development experiments and the manuscript draft; LG Mesquita contributed with the TUNEL experiments and drafting the article; MR Chiaratti contributed with the qPCR experiments and data analysis; FV Meirelles contributed with the design of the study and the manuscript draft; and CLV Leal contributed with the design of the study and the manuscript draft.
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Non-invasive Methods to Enhance in vitro Embryo Production
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Combination of oocyte and zygote selection by brilliant cresyl blue (BCB) test enhanced prediction of developmental potential to the blastocyst in cattle. Anim Reprod Sci 136, 245–251. Mossa F, Walsh SW, Butler ST, Berry DP, Carter F, Lonergan P, Smith GW, Ireland JJ, Evans AC, 2012: Low numbers of ovarian follicles >/=3 mm in diameter are associated with low fertility in dairy cows. J Dairy Sci 95, 2355–2361. Opiela J, Katska-Ksiazkiewicz L, Lipinski D, Slomski R, Bzowska M, Rynska B, 2008: Interactions among activity of glucose-6-phosphate dehydrogenase in immature oocytes, expression of apoptosis-related genes Bcl-2 and Bax, and developmental competence following IVP in cattle. Theriogenology 69, 546– 555. Pancarci SM, Ari UC, Atakisi O, Gungor O, Cigremis Y, Bollwein H, 2012: Nitric oxide concentrations, estradiol-17beta progesterone ratio in follicular fluid, and COC quality with respect to perifollicular blood flow in cows. Anim Reprod Sci 130, 9–15. Parrish JJ, Susko-Parrish J, Winer MA, First NL, 1988: Capacitation of bovine sperm by heparin. Biol Reprod 38, 1171– 1180. Pavlok A, Lucas-Hahn A, Niemann H, 1992: Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles. Mol Reprod Dev 31, 63–67. Pfaffl MW, Horgan GW, Dempfle L, 2002: Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30, e36. Pocar P, Nestler D, Risch M, Fischer B, 2005: Apoptosis in bovine cumulus-oocyte complexes after exposure to polychlorinated biphenyl mixtures during in vitro maturation. Reproduction 130, 857–868. Pontes JH, Melo Sterza FA, Basso AC, Ferreira CR, Sanches BV, Rubin KC,
Seneda MM, 2012: Ovum pick up, in vitro embryo production, and pregnancy rates from a large-scale commercial program using Nelore cattle (Bos indicus) donors. Theriogenology 75, 1640–1646. Rizos D, Lonergan P, Boland MP, Arroyo-Garcia R, Pintado B, de la Fuente J, Gutierrez-Adan A, 2002: Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: implications for blastocyst quality. Biol Reprod 66, 589– 595. Rizos D, Clemente M, Bermejo-Alvarez P, de La Fuente J, Lonergan P, Gutierrez-Adan A, 2008: Consequences of in vitro culture conditions on embryo development and quality. Reprod Domest Anim 43(Suppl 4), 44–50. Sananmuang T, Tharasanit T, Nguyen C, Phutikanit N, Techakumphu M, 2011: Culture medium and embryo density influence on developmental competence and gene expression of cat embryos. Theriogenology 75, 1708–1719. dos Santos LG, Lopes-Costa PV, dos Santos AR, Facina G, da Silva BB, 2008: Bcl-2 oncogene expression in estrogen receptor-positive and negative breast carcinoma. Eur J Gynaecol Oncol 29, 459–461. Su J, Wang Y, Li R, Peng H, Hua S, Li Q, Quan F, Guo Z, Zhang Y, 2012: Oocytes selected using BCB staining enhance nuclear reprogramming and the in vivo development of SCNT embryos in cattle. PLoS ONE 7, e36181. Sutton-McDowall ML, Mottershead DG, Gardner DK, Gilchrist RB, Thompson JG, 2012: Metabolic differences in bovine cumulus-oocyte complexes matured in vitro in the presence or absence of follicle-stimulating hormone and bone morphogenetic protein 15. Biol Reprod 87, 87. Takase K, Ishikawa M, Hoshiai H, 1995: Apoptosis in the degeneration process of unfertilized mouse ova. Tohoku J Exp Med 175, 69–76.
Tanghe S, Van Soom A, Mehrzad J, Maes D, Duchateau L, de Kruif A, 2003: Cumulus contributions during bovine fertilization in vitro. Theriogenology 60, 135– 149. Van Soom A, Tanghe S, De Pauw I, Maes D, de Kruif A, 2002: Function of the cumulus oophorus before and during mammalian fertilization. Reprod Domest Anim 37, 144–151. Vogel MW, 2002: Cell death, Bcl-2, Bax, and the cerebellum. Cerebellum 1, 277–287. Wasielak M, Bogacki M, 2007: Apoptosis inhibition by insulin-like growth factor (IGF)-I during in vitro maturation of bovine oocytes. J Reprod Dev 53, 419– 426. de Wit AA, Kruip TA, 2001: Bovine cumulus-oocyte-complex-quality is reflected in sensitivity for alpha-amanitin, oocytediameter and developmental capacity. Anim Reprod Sci 65, 51–65. Yang MY, Rajamahendran R, 2002: Expression of Bcl-2 and Bax proteins in relation to quality of bovine oocytes and embryos produced in vitro. Anim Reprod Sci 70, 159–169. Yuan YQ, Van Soom A, Leroy JL, Dewulf J, Van Zeveren A, de Kruif A, Peelman LJ, 2005: Apoptosis in cumulus cells, but not in oocytes, may influence bovine embryonic developmental competence. Theriogenology 63, 2147–2163.
Submitted: 28 Aug 2013; Accepted: 3 Apr 2014 Author’s address (for correspondence): THC De Bem, ZAB-FZEA-USP, Av. Duque de Caxias Norte, 225, Pirassununga, SP 13635900, Brazil. E-mail: tiagodebem@yahoo. com.br
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The Influence of Morphology, Follicle Size and Bcl-2 and Bax Transcripts on the Developmental Competence of Bovine Oocytes THC De Bem1,2, PR Adona3,4, FF Bressan1, LG Mesquita1, MR Chiaratti5, FV Meirelles1 and CLV Leal1 1
Departamento de Ci^ encias, Faculdade de Zootecnia e Engenharia de Alimentos B asicas, Universidade de S~ ao Paulo, Pirassununga, Brazil; Departamento de Gen etica, Faculdade de Medicina de Ribeir~ ao Preto, Universidade de S~ ao Paulo, Ribeir~ ao Preto, Brazil; 3Universidade Norte do aria Laffranchi, Tamarana, Brazil; 5Departamento de Gen etica e Evolucßa~o, Universidade Federal de S~ ao Paran a, Londrina, Brazil; 4Agropecu Carlos, S~ ao Carlos, Brazil 2
Contents This study analysed two non-invasive oocyte selection methods in relation to in vitro embryo development capacity and expression of apoptosis-related genes. Selection was based on morphological quality of oocytes or follicle diameter. Oocytes were classified as grade I (GI ≥3 layers compact cumulus cells and homogeneous cytoplasm; grade II (GII ≤3 layers compact cells and homogeneous cytoplasm;, and grade III (GIII ≥3 layers, but cells with slight expansion and slightly granulated cytoplasm). Blastocyst development was lower for GII (28.5%) than for GIII (47.7%, p < 0.05), and GI was similar to both (36.9%, p > 0.05). Relative expression of Bcl-2 gene was lower in the GI (1.0, p < 0.05) than in the GII (1.8) and GIII (2.2), which were not different (p > 0.05). There was no difference (p > 0.05) between GI (1.0), GII (0.92) and GIII (0.93) regarding the Bax transcript. However, the Bax and Bcl-2 transcript ratios in GII (Bax; 0.92 and Bcl-2; 1.8) and GIII (Bax; 0.93 and Bcl-2; 2.2) were different (p < 0.05). Regarding oocytes from follicles of different sizes, cleavage and blastocyst rates for 1–3 mm (82.5; 23.7%) were lower (p < 0.05) than for 6–9 mm (95.6; 41.1%), but similar (p > 0.05) to 3–6 mm (93.7; 35.4%), which were not different (p > 0.05). Regarding Bax and Bcl-2 expression, the oocytes were similar (p > 0.05) for 1–3 mm (Bax; 1.0 and Bcl-2; 1.0), 3–6 mm (Bax; 1.0 and Bcl-2; 0.93) and 6–9 mm (Bax; 0.92 and Bcl-2; 0.91). In conclusion, oocyte selection based on morphological appearance does not guarantee the success of embryonic development. Additionally, the absence of apoptosis is not necessarily a benefit for the development of oocytes. Bovine COCs with initial signs of atresia may be used for the in vitro production of embryos, and COCs taken from follicles >3 mm in diameter are better suited to in vitro embryo development.
Introduction The in vitro production of embryos is an important biotechnological technique in the field of animal reproduction, widely used in research, but also in commercial settings. Notwithstanding its widespread use, the in vitro production of embryos associated with follicular aspiration guided by ultrasound (ovum pickup) (Pontes et al. 2012) or ovaries taken from abattoirs, an abundant source of oocytes (Dey et al. 2012), still suffers certain limitations due to the relatively low production of embryos (Rizos et al. 2008; Adona et al. 2011). Several analyses have focused on the interaction between developmental competence and different oocyte characteristics. These include studies focusing on the morphology of cumulus–oocyte complexes (Wasielak and Bogacki 2007; Alvarez et al. 2009; Melka et al. 2010),
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the diameter of the oocyte or the size of the follicle from which they are aspirated (Kauffold et al. 2005; Han et al. 2006), the follicular wave phase (Blondin and Sirard 1995; Mossa et al. 2012; Pancarci et al. 2012), the communication of the oocyte with the cumulus cells (Krisher 2004), the accumulated amount of mRNA among other molecules which have been shown to influence, although to different degrees, the growth and potential of the oocyte to develop to the blastocyst stage (Meirelles et al. 2004; Adona et al. 2011; Boumela et al. 2011; SuttonMcDowall et al. 2012). The selection of oocytes based on morphology was first studied in cattle by Leibfried and First (1979); since then, the cumulus–oocyte complexes used in the in vitro production of embryos of several species are usually selected according to the morphological characteristics of the cells and the appearance of the ooplasm under a stereomicroscope (Leon et al. 2013). However, even when using cumulus–oocyte complexes (COCs) regarded as of high quality using this methodology, such COCs do not necessarily possess the best development capacity (Blondin and Sirard 1995; Li et al. 2009). Together with the morphological selection of cumulus–oocyte complexes, the diameter of the follicle is one of the most commonly used parameters in the prediction of bovine oocyte competence (Anguita et al. 2007). The relationship between the diameter of the follicle and the developmental competence of the oocyte has already been shown (Pavlok et al. 1992), whereby some studies indicate a positive relationship between the diameter of the follicle and the in vitro blastocyst developmental competence (Blondin et al. 1997; Kauffold et al. 2005; Han et al. 2006). Oocytes from follicles smaller than 3 mm in diameter are less competent for the in vitro production embryos when compared with those from larger follicles (Lonergan et al. 1994; Kauffold et al. 2005). However, the diameter does not necessarily indicate whether the follicle is healthy or is already in a state of atresia. The selection of oocytes based on morphology and follicle diameter is a common practice in the in vitro production of embryos, but the effectiveness in determining post-fertilization embryo competence is still uncertain (Ebner et al. 2003). In turn, it is known that the presence of programmed cell death may lead to DNA fragmentation and oocyte degeneration (Takase et al. 1995; Matwee et al. 2000), meaning a significant decrease in fertility of oocytes used for the in vitro production of embryos (Fujino et al. 1996).
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THC De Bem, PR Adona, FF Bressan, LG Mesquita, MR Chiaratti, FV Meirelles and CLV Leal
The Bcl-2 gene family, which includes the Bax (proapoptotic) and Bcl-2 (anti-apoptotic) genes, is involved in the regulation of apoptosis (Yang and Rajamahendran 2002). These two genes in particular play a key role in the occurrence of apoptosis in female germ cells (Kim and Tilly 2004) and are used in the analysis of apoptosis in oocytes and embryos (Yang and Rajamahendran 2002; Pocar et al. 2005; Opiela et al. 2008). Research assessing the developmental competence of oocytes selected according to morphology is quite inconsistent and even contradictory (Yuan et al. 2005; Li et al. 2009). Some demonstrate that COCs with early signs of atresia (e.g. a slight increase in the size of the cumulus and a slight cytoplasmic granulation) show greater developmental potential than those regarded as morphologically healthy (Blondin and Sirard 1995; Alvarez et al. 2009; Li et al. 2009). Most studies on in vitro production of embryos is performed only with grade I oocytes obtained from follicles measuring from 3 to 8 mm in diameter, but we believe that a considerable number of oocytes, which do not strictly fall within this classification standard (Grade I), could still be used in the IVP system. With the aim of maximizing the use of oocytes for the in vitro production of embryos, the purpose of this study was to analyse two non-invasive (morphological quality and follicle size) methods and one invasive (genes related to apoptosis – Bcl-2 and Bax) method in the selection of oocytes, associating them with developmental competency and the presence of DNA fragmentation in the embryos to better understand the role of the origin and oocyte quality on development in vitro and quality of blastocysts.
Material and Methods Chemicals were acquired from the Sigma Chemical Company, St Louis, MO, USA, unless specified otherwise. Selection of cumulus–oocyte complexes (COCs) and follicle classification Bovine ovaries were collected from a commercial abattoir and transported to the laboratory in saline (NaCl 0.9%) with antibiotics (50 lg/ml gentamicin) at 25– 30°C. The ovaries were aspirated (Adona et al. 2008a,b) and the COCs separated according to morphological classes: grade I (GI) with more than three layers of compact cells and a homogeneous cytoplasm; grade II (GII) with up to three layers of compact cells and a homogeneous cytoplasm; and grade III (GIII) with more than three layers of cells with a slight expansion and a slightly granulated cytoplasm (de Loos et al. 1992). COCs were also collected according to follicle diameter. For this purpose, the follicles located in the ovarian cortical region were measured with a calliper and classified in different sizes (1–3, 3–6 and 6–9 mm in diameter). From the different follicles sizes, only grade I COCs were used.
In vitro maturation The COCs selected for the experiments were transferred to the maturation medium [199 medium supplemented with 10% foetal bovine serum, 5.0 lg/ml luteinizing hormone (Lutropin-v, Vetrepharm), 0.5 lg/ml folliclestimulating hormone (Folltropin-v, Vetrepharm), 0.2 mM pyruvate and 50 lg/ml gentamicin]. In vitro maturation was performed for 22 h in 100 ll droplets ( 20 COCs per drop) under mineral oil at 38.5°C and an atmosphere of 5% CO2 in air. In vitro fertilization and culture COCs matured in vitro were fertilized with the thawed semen (Genetica Avancßada, S~ao Carlos, Brazil) from the same bull and batch and prepared according to the Percoll gradient technique (Amersham Pharmacia Biotec). Fertilization (2 9 106 spermatozoa/ml) was conducted in TALP medium (Parrish et al. 1988), supplemented with 2 lM penicillamine, 1 lM hypotaurine, 250 lM epinephrine and 20 lg/ml heparin. COCs and spermatozoa were incubated in 100 ll droplets under mineral oil for 18 h. After this period, the COCs were partially denuded and transferred to an in vitro synthetic oviductal fluid culture medium (Holm et al. 1999) supplemented with 2.5% foetal calf serum, 0.2 mM pyruvate and 50 lg/ml gentamicin. Cleavage rate was analysed after 48 h of culture (D2), blastocyst rate on the seventh day (D7) and blastocyst hatching rate on the ninth day (D9). In vitro culture of embryos was performed at 38.5°C and an atmosphere of 5% CO2 in air. Detection of nuclear fragmentation using the TUNEL technique Hatched embryos (D9) were placed in 3% paraformaldehyde and permeabilized in a PBS solution supplemented with polyvinyl alcohol (PVA), 0.5% Triton X100 (USB) and 0.1% sodium citrate for 1 h at room temperature. The TUNEL technique was conducted using the ‘In Situ Cell Death Detection Kit Fluorescein’ (Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. The embryos were separated in groups for negative control analysis (absence of stained cells), positive control analysis (stained cells) and experimental samples. The analyses were performed under an epifluorescent microscope; the cells stained in green (FITC) were considered as positive TUNEL cells, that is, with fragmented DNA, and a blue stain (Hoechst 33342) indicated the location of the nucleus of the cells. Extraction of RNA and reverse transcription (cDNA) After being selected and denuded, the oocytes were stored at 80°C in calcium- and magnesium-free PBS with 0.1% PVA and 100 Ul/ml RNAse inhibitor (Life Technologies, Grand Island, NY, USA). RNA was extracted from pools of 25 oocytes from each group using an RNeasy Protect Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcription (cDNA) of RNA was performed
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Non-invasive Methods to Enhance in vitro Embryo Production
using an oligo (dT) 12–18 primer according to the manufacturer’s recommendations (Improm-II; Promega, Madison, WI, USA). Samples (20 ll) were then frozen for real-time PCR analysis. Quantitative PCR Relative real-time PCR quantification and amplification of cDNA was performed using the kit Power SYBR Green PCR Master Mix according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA) in triplicate. The thermal cycling conditions included initial sample incubation at 50°C for 2 min and at 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and at 60°C for 45 s using an ABI 7500 instrument (Applied Biosystems). Standard curves were generated for each gene (Bax, Bcl-2 and GAPDH – Table 1) by serial dilution using fivefold dilutions (1:3) to create a 5-point standard curve, and the slope was between 3.3 and 3.6, with an R2 > 0.95. Standard curves were performed in a 20 ll reaction volume [10 ll SYBR Green PCR master mix, 1 ll of each primer (10 lM), 7 ll of nuclease-free water and 2 ll cDNA]. In each sample, the median value of PCR triplicates was considered, and Bax and Bcl-2 data were normalized according to the relative concentration of the expression normalizer, the endogenous 3-phosphate dehydrogenase gene (GAPDH). The calculations of relative quantification were conducted using the comparative cycle threshold (Ct) method, and the PCR product was analysed using a dissociation curve.
Experimental Design Experiment 1: the influence of COCs morphology on the in vitro production of embryos The COCs aspirated from 2 to 8 mm follicles were classified under a stereomicroscope and divided into the three morphological (I, II and III). Subsequent to classification, they were matured (22 h), fertilized (18 h) and cultured in vitro for 9 days (D9 = 216 h post-fertilization) for the analysis of blastocyst, hatching and apoptosis rates. Four replicates were performed for analyses of embryo development. Three replicates were performed for the analysis of DNA fragmentation, and the blastocysts (D9) were analysed under an epifluorescent microscope for the quantification of apoptotic cells (TUNEL+). Experiment 2: the influence of follicle size on the in vitro production of embryos The COCs aspirated from follicles of different diameters (1–3, 3–6 and 6–9 mm) were selected under a
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stereomicroscope, and all the viable oocytes (not degenerated or denuded) were matured (22 h), fertilized (18 h) and cultured in vitro for up to 9 days (D9) for the analysis of the blastocyst development, hatching and apoptosis rates. Four replicates were performed for analyses of embryo development. Three replicates were performed for the analysis of DNA fragmentation, and the blastocysts (D9) were analysed under an epifluorescent microscope for the quantification of apoptotic cells (TUNEL+). Experiment 3: relative expression of Bax and Bcl-2 genes in bovine oocytes from COCs of different morphological qualities and from different follicle diameters The immature COCs were classified according to morphology (GI, GII and GIII) and the diameter of the follicle (1–3, 3–6 and 6–9 mm in diameter). After being classified, they were denuded, split into pools of 25 oocytes for each group in four replicate batches and stored at 80°C. The analysis of the transcripts involved the extraction of the RNA, reverse transcription (cDNA) and the relative quantification of the genes in real-time PCR (Fig. 1). Statistical analysis The variables for cleavage, blastocysts and hatching were transformed according to the arcsin function. Nontransformed numbers were used for the analysis of total cell numbers and TUNEL+ cells. The BIOESTATS 5.0 (Instituto de Desenvolvimento Sustentavel Mamirau a, Tefe, AM, Brazil) program was used for all the analyses, and ANOVA was applied at 5% significance to reveal the differences between the treatments. The Tukey post hoc test was used for significant results in the analysis of variances as a multiple comparison procedure between the groups. The relative quantification of the real-time PCR transcripts was analysed using the 2005 – Beta V19.9 REST (Relative Expression Software Tool; Pfaffl et al. 2002) at a significance level of 5% to reveal the differences between transcripts (www.gene-quantifi cation-info).
Results Experiment 1 COCs from different morphological categories were subjected to fertilization for the analysis of in vitro embryo development and the percentage of TUNEL+ cells (Tables 2 and 3). The cleavage rate was approximately 82% and was similar (p > 0.05) for all COCs
Table 1. Sequences of the gene primers analysed in the different treatments Genes
Primers
Bp
Gene bank
GAPDH
F:50 -CGACTTCAACAGCGACACTCA-30 R:50 -AGCCAAATTCATTGTCGTACCA-30 F:50 -TTTGCTTCAGGGTTTCATCCA-30 R:50 -CCGATGCGCTTCAGACACT-30 F:50 -GAGTCGGATCGCAACTTGGA-30 R:50 -CTCTCGGCTGCTGCATTGT-30
107
NM_001034034.2
126
NM_173894.1
120
NM_001077486.2
BAX BCL2
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THC De Bem, PR Adona, FF Bressan, LG Mesquita, MR Chiaratti, FV Meirelles and CLV Leal
Table 2. Embryo development and blastocyst hatching rates according to morphological classification of COCs
Group GI GII GIII
Oocytes N
Cleavage D2 n (% SD)
Blastocysts D7 n (% SD)
Hached D9 n (% SD)
92 91 88
78 (85 4.0) 73 (80 8.8) 72 (82 4.2)
34 (36.9 1.1)ab 26 (28.5 4.8)b 42 (47.7 5.5)a
31 (91.2 5.2) 22 (84.6 8.3) 37 (88.1 8.4)
SD, standard deviation of the mean. Results of four replicates. Different letters within the same column indicate a difference between treatments.
Table 3. Total cell numbers and the percentage of positive TUNEL cells in the embryos from COCs of different morphological categories
Group
Hached D9 N
Cell numbers n ( SD)
TUNEL+ cells% ( SD)
16 16 18
177 ( 29.6) 205 ( 25.9) 185 ( 33.6)
0.14 ( 0.02) 0.32 ( 0.28) 0.84 ( 0.68)
GI GII GIII
SD, standard deviation of the mean. Results of four replicates.
Table 4. Analysis of the COCs taken from follicles of different sizes in relation to embryo development and blastocyst hatching
Group 1–3 mm 3–6 mm 6–9 mm
Oocytes N
Cleavage D2 n (% SD)
Blastocysts D7 n (% SD)
Hached D9 n (% SD)
80 79 68
66 (82.5 3.5)b 74 (93.7 3.0)ab 65 (95.6 9.5)a
19 (23.7 4.7)b 28 (35.4 6.3)ab 28 (41.2 2.8)a
15 (78.9 11.0) 22 (78.6 11.6) 21 (75.0 10.7)
SD, standard deviation of the mean. Results of four replicates. Different letters within the same column indicate a difference between treatments.
groups analysed. The only difference (p < 0.05) detected in relation to in vitro embryo development (D7 blastocysts) was between the GII (28.5%) and GIII (47.7%) groups. The GI group (36.9%) did not differ from the others (p > 0.05). There were also no differences (p > 0.05) in the percentage of hatched blastocysts (D9) for GI (91.2%), GII (84.6%) and GIII (88.1%) COCs; in the average total cell numbers (177, 205 and 185 for GI, GII and GIII, respectively); and in the percentage of positive TUNEL cells (0.14, 0.32 and 0.84%, for GI, GII and GIII, respectively) in hatched embryos.
Bax
(a)
Bcl-2
(b)
2.0
1.0
0.0
a a
a b
a b
GI
GII
GIII
Cumulus oocyte complex morphogy
Relative expression
Relative expression
3.0
Bax
3.0
Table 5. Analysis of the number of cells and the percentage of positive TUNEL cells in the embryos of COCs from different-sized follicles
Group
Hached D9 N
Cell numbers n ( SD)
TUNEL+ cells % ( SD)
15 16 16
268 ( 22.2) 248 ( 17.7) 237 ( 12.1)
0.36 ( 0.18) 0.30 ( 0.17) 0.23 ( 0.27)
1–3 mm 3–6 mm 6–9 mm
SD, standard deviation of the mean. Results of four replicates.
Experiment 2 The COCs were aspirated from follicles of different diameters for the analysis of in vitro embryo development and the percentage of TUNEL+ cells (Tables 4 and 5). The cleavage and blastocyst rates on day seven in the 1–3 mm group (82.5; 23.7%) were lower (p < 0.05) in relation to the 6–9 mm group (95.6; 41.1%), but did not differ (p > 0.05) from 3 to 6 mm (93.7; 35.4%), and these groups did not differ between themselves (p > 0.05). There was also no difference (p > 0.05) in the hatching rate of the COCs from the 1 to 3 mm (78.9%), 3 to 6 mm (78.6%) and 6 to 9 mm (75.0%) groups. The average number of cells (237 to 268) in the embryos and the percentage of apoptotic cells (TUNEL+) for the 1–3 mm (0.36%), 3–6 mm (0.30%) and 6–9 mm (0.23%) groups did not differ (p > 0.05). Experiment 3 Oocytes with different morphological qualities taken from follicles of different diameters were evaluated for relative quantification of Bax and Bcl-2 transcripts. There was a difference (p < 0.05) in the relative expression of the Bcl-2 gene between the GI (1.0) group and the GII (1.8) and GIII (2.2) groups, but no such difference between the GII and GIII groups (p > 0.05) (Fig. 1a). There was also no difference (p > 0.05) between the GI (1.0), GII (0.92) and GIII (0.93) groups for relative expression of the Bax transcript (Fig. 1a). There was difference (p < 0.05) between the two Bax and Bcl-2 transcripts only in the GII (Bax = 0.92 and Bcl-2 = 1.8) and the GIII groups (Bax = 0.93 and Bcl-2 = 2.2) (Fig. 1a). For oocytes from follicles of different sizes (Fig. 1b), the relative expression of Bax and Bcl-2 genes did not vary (p > 0.05) between groups 1–3 mm (Bax = 1.0 and Bcl-2 = 1.0), 3–6 mm (Bax = 1.0 and
Bcl-2
2.0
1.0
0.0 1–3
3–6
6–9
Oocytes from follicles of different sizes
Fig. 1. Relative expression of Bax and Bcl-2 genes in oocytes separated by morphological criteria (a) and oocytes taken from different-sized follicles (b)
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Non-invasive Methods to Enhance in vitro Embryo Production
Bcl-2 = 0.93) and 6–9 mm (Bax = 0.92 and Bcl2 = 0.91), or even between the two (Bax and Bcl-2) genes.
Discussion In the majority of studies, bovine oocytes are considered to be of good quality when they have various compact layers of cumulus cells and homogenous cytoplasm (Wasielak and Bogacki 2007), as the physiological state of the cumulus cells indicated by morphological and physical differences increases the rate of post-fertilization maturation and development (Blondin and Sirard 1995; de Wit and Kruip 2001; Boni et al. 2002). The maturation and posterior development of the oocyte are partially dependent on cumulus cells, which are highly specialized cells and have a transzonal cytoplasmic structures that penetrates the zona pellucida and touches the plasma membrane of the oocyte (Albertini et al. 2001), creating the cumulus oocyte complex (COC). Previous studies examining the embryonic development capacity of oocytes, based on visual appearance, reported that COCs with early signs of atresia (partial expansion of cumulus and small granules in cytoplasm) possess greater embryonic growth potential than COCs that were considered morphologically healthy and of higher quality (de Wit and Kruip 2001; BilodeauGoeseels and Panich 2002; Li et al. 2009). In the present study, the separation of COCs by morphological criteria into GI, GII and GIII groups showed that embryonic development may be affected by the morphology of the oocytes, as the GIII group, considered to be of poor morphological quality, was superior to the intermediate group (GII), but similar to the GI group. However, when the number of cells in the hatched blastocysts and the percentage of positive TUNEL cells were evaluated, no differences were observed between the groups (GI, GII and GIII). Although there was no reduction in the cleavage rate, the percentage of embryos that reached the blastocyst stage was therefore affected by the morphological classification of the COCs, which is in accordance with previous findings (Boni et al. 2002; Li et al. 2009). These results confirm that morphological classification can affect the proportion of blastocysts formed, but not their quality, as COCs classified as GI, GII and GIII did not reveal differences in the number of blastocyst cells and the percentage of positive TUNEL cells. Melka et al. (2010) found few TUNEL+ cells in good quality embryos. This suggests that in this study the embryos from oocytes from GI, GII and GIII groups are of good quality, due to the total number of cells and the low percentage of TUNEL+ cells, and also that the grade of the oocyte does not interfere with such criteria. From this assumption, it may be suggested that the percentage of TUNEL+ cells (6 mm), and these were similar to those from follicles of intermediate diameter (3–6 mm). These results are similar to those reported (Lonergan et al. 1994; Gandolfi and Gandolfi 2001; Kauffold et al. 2005) which state that follicles with diameter >3 mm contain COCs with higher development potential than smaller follicles and the quality of those embryos is closer to those from an in vitro culture system (Rizos et al. 2002; Cui et al. 2011; Sananmuang et al. 2011) as previously mentioned in the present study. The removal of COCs from follicles smaller than 3 mm in diameter before oocyte capacitation is finalized (Hyttel et al. 1996) is, probably, one of the factors involved in the low production of embryos in vitro. Therefore, the interruption of folliculogenesis is responsible for the reduction in oocyte competence. In bovine and ovine, COCs from follicles smaller than 3 mm in diameter have oocytes with a smaller diameter, while follicles >3 mm in diameter have oocytes with a larger diameter (Mirshamsi et al. 2013). The diameter of the follicles affects the expansion of the cumulus cells, in vitro maturation and in the embryonic development of COCs in cattle and sheep (Kauffold et al. 2005; Han et al. 2006). It was also found in sheep that COCs from follicles with diameters smaller than 3 mm had brilliant cresyl blue staining (BCB) inferior to COCs from larger follicles (Mirshamsi et al. 2013), indicating that these COCs are less developmentally competent. Brilliant Cresyl blue staining has been used to evaluate the quality of COCs and their developmental competence (Janowski et al. 2012; Su et al. 2012). In the evaluation of Bcl-2 and Bax transcripts from follicles with different diameters, the expression of these genes and the ratio between them were not affected. Evans et al. (2004) analysed apoptotic genes in granulosa and theca cells during the follicular waves in cows and did not find differences in the ratio of expression of Bcl-2 and Bax between the dominant and subordinate follicles. In another study, the Bcl-2 transcript abundance in GV (germinal vesicle) oocytes was not significantly affected by diameter of the follicle from which the oocytes were derived, but after the IVM culture, the Bcl-2 transcript abundance in MII (metaphase-II) oocytes was significantly affected (Kohata et al. 2013). The transcription abundance of Bcl-2 gene is gradually reduced during ageing of oocytes, resulting in decreasing anti-apoptotic protein Bcl-2 and leading to abnormal sister chromatid segregation during meiosis II, embryo fragmentation and apoptosis (Kohata et al. 2013). Therefore, we believe that the differential expression of the apoptotic genes Bcl-2 and Bax is related only in oocytes with advanced signs of atresia. In this way,
the follicles used during these experiments were likely collected before or immediately after the selection of the dominant follicle and prior to both DNA fragmentation and global activation of the genes involved in the terminal stages of apoptosis (Austin et al. 2001; Evans et al. 2004). The expression of Bax may be constitutive, which suggests that the oocytes are under constant threat and that their development potential depends on their capacity to inhibit pro-apoptotic activity (Boumela et al. 2011). This suggests that changes in gene expression itself are not sufficient to prevent apoptosis. The balance between Bcl-2/Bax in oocytes from differentsized follicles suggests that they have undergone similar development. However, there was a reduction in the level of embryos in the group of COCs from small follicles, but this reduction in the production of embryos may be related to other factors, as previously mentioned in the present study. In conclusion, oocyte selection based on morphological appearance does not guarantee the success of embryonic development. Bovine COCs with initial signs of atresia may be used for the in vitro production of embryos, and COCs from follicles >3 mm in diameter are more suitable for in vitro embryo development than oocytes from smaller follicles. Additionally, the absence of apoptosis is not necessarily a benefit for the development of oocytes; therefore, a moderate apoptosis of the blastocyst may not be harmful for pre-implantational development. The evaluation of Bcl-2 and Bax transcripts is not determinative as biomarkers of the quality of the oocyte and its pre-implantation developmental potential. This suggests that the developmental potential of an oocyte is dependent on numerous variables, and multiple factors must be considered to determine the quality of an oocyte. However, it would be interesting to analyse the blastocysts transfers to observe the relationship between oocyte quality, follicle size and the apoptosis levels with the pregnancy rates and the success of development to term. Acknowledgements The present study was supported by FAPESP, Brazil (Grant # 05/ 59694-5). THCDB and PRA were supported by FAPESP, Brazil (06/ 58536-0 and 07/51953-9, respectively).
Conflict of interest None of the authors have any conflict of interest to declare.
Author contributions THC De Bem contributed with all the in vitro development experiments, carrying out the experiments, data analysis and drafting the paper; PR Adona contributed with the experimental design, carrying out the experiments and drafting the article; FF Bressan contributed with in vitro development experiments and the manuscript draft; LG Mesquita contributed with the TUNEL experiments and drafting the article; MR Chiaratti contributed with the qPCR experiments and data analysis; FV Meirelles contributed with the design of the study and the manuscript draft; and CLV Leal contributed with the design of the study and the manuscript draft.
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Non-invasive Methods to Enhance in vitro Embryo Production
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Submitted: 28 Aug 2013; Accepted: 3 Apr 2014 Author’s address (for correspondence): THC De Bem, ZAB-FZEA-USP, Av. Duque de Caxias Norte, 225, Pirassununga, SP 13635900, Brazil. E-mail: tiagodebem@yahoo. com.br
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