Introduction
New knowledge about developmental competence, in vitro maturation (IVM) and cryopreservation of oocytes and embryos is critical for the in vitro production (IVP) of embryos. The techniques of assisted reproduction, in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) have been successfully performed with equine gametes, however low efficiency has limited their commercial application in this species (Squires et al., Reference Squires, Carnevale, McCue and Bruemmer2003; Choi et al., Reference Choi, Love, Varner and Hinrichs2006; Hinrichs et al., Reference Hinrichs, Choi, Walckenaer, Varner and Hartman2007). Progress in this area was initially slow as standard IVF is not reproducible in horses (Hinrichs, Reference Hinrichs2010). However, ICSI resulted in the birth of several foals, and has been used for the clinical production of foals (Galli et al., Reference Galli, Colleoni, Duchi, Lagutina and Lazzari2007; Jacobson et al., Reference Jacobson, Choi, Hayden and Hinrichs2010; Mortensen et al., Reference Mortensen, Choi, Ing, Kraemer, Vogelsang and Hinrichs2010). Nevertheless, oocyte quality and culture conditions were found to be highly variable, with low levels of blastocyst formation in vitro and poor reproducibility between laboratories (Dell'Aquila et al., Reference Dell'Aquila, Albrizio, Maritato, Minoia and Hinrichs2003; Smits et al., Reference Smits, Goossens, Van, Govaere, Hoogewijs, Vanhaesebrouck, Galli, Colleoni, Vandesompele and Peelman2009).
The study of gene expression is emerging in applied embryology, those genes involved in biological processes such as apoptosis, oxidative stress and cryotolerance need to be studied in order to compare gene expression in vivo, in vitro and under different culture conditions (Wrenzycki et al., Reference Wrenzycki, Herrmann, Lucas-Hahn, Korsawe, Lemme and Niemann2005; Badr et al., Reference Badr, Bongioni, Abdoon, Kandil and Puglisi2007; Rizos et al., Reference Rizos, Clemente, Bermejo-Alvarez, de La, Lonergan and Gutierrez-Adan2008). Oocyte quality can be assessed immediately following recovery by using several non-invasive, visual assessment parameters such as the morphology of the cumulus–oocyte complexes (COC). Oocytes with a compact cumulus composed of several layers of cells and a homogeneous cytoplasm are considered to be healthy; these oocytes can then be selected for IVM and IVF (Li et al., Reference Li, Liu, Cang, Wang, Jin, Ma and Shorgan2009). However, it has been reported that equine oocytes with a compact cumulus exhibited a lower meiotic competence and lower fertilization rate after ICSI (Ambruosi et al., Reference Ambruosi, Lacalandra, Iorga, De, Mugnier, Matarrese, Goudet and Dell'Aquila2009). Analysis of the expression of genes that are involved in early embryonic development is an important tool for the development of assisted reproduction biotechnology.
One of the main factors affecting the potential for embryonic development is apoptosis (Park et al., Reference Park, Kim, Cui, Tae, Lee, Kim, Park and Lim2006; Dhali et al., Reference Dhali, Anchamparuthy, Butler, Pearson, Mullarky and Gwazdauskas2007; Anguita et al., Reference Anguita, Paramio, Morato, Romaguera, Jimenez-Macedo, Mogas and Izquierdo2009), or programmed cell death, which is an active and irreversible process of self-destruction of cells under physiological control (Parolin & Reason, Reference Parolin and Reason2001). The mechanisms of apoptosis may be activated by external stimuli, by binding to specific receptors on the cell surface, or by internal stimuli to intracellular stress, such as DNA damage, cell cycle alterations and in metabolic pathways and is controlled by several different gene families (Chang et al., Reference Chang, Cvetanovic, Harvey, Komoriya, Packard and Ucker2002). Previous studies on cumulus cell gene expression (McKenzie et al., Reference McKenzie, Pangas, Carson, Kovanci, Cisneros, Buster, Amato and Matzuk2004) and apoptosis (Corn et al., Reference Corn, Hauser-Kronberger, Moser, Tews and Ebner2005) showed that cumulus cells can reflect the developmental potential of human embryos during IVF cycles. Previously, the apoptosis incidence in mare ovarian follicles, its relationship with cumulus expansion and increased meiotic competence was demonstrated, however, no association with cytoplasmic maturation was observed (Dell'Aquila et al., Reference Dell'Aquila, Albrizio, Maritato, Minoia and Hinrichs2003). The impact of apoptosis in the COC and its impact on oocyte development potential remains unclear, as contradictory reports from various species have failed to clarify the occurrence of apoptosis in the COC (Yuan et al., Reference Yuan, Van, Leroy, Dewulf, Van, de and Peelman2005).
Thus, the aim of the current study was to evaluate the expression of the apoptosis-related genes Bax, Bcl-2, survivin and p53 during IVM of equine oocytes and to compare gene expression between oocytes and cumulus cells isolated from COC with different morphological characteristics.
Materials and methods
Cumulus–oocyte complexes (COC) source
The biological material used in this experiment was obtained from a horse abattoir located in the city of Pelotas, RS, Brazil. The ovaries were collected randomly on the slaughter line, without identifying age, stage of the estrous cycle, clinical condition and nutritional status of mares. The time between slaughter and the collection was approximately 1 h, and the ovaries were transported in thermobottles in 0.9% NaCl sterile solution at 32–35°C to the Laboratory of Molecular Embryology, UFPel. The COC were aspirated from follicles ranging from 10 to 20 mm in diameter (follicles preceding follicle deviation). The contents were placed into a 50 ml conical tube and allowed to settle for 15 min. The sediment was evaluated using a stereomicroscope (Olympus) and suitable COC were selected. Degenerated or metaphase II (MII) stage oocytes were discarded.
Morphological characterization of the equine COC
The COC were evaluated morphologically using an inverted optical microscope (Olympus) for the number of layers and degree of compaction of the cumulus cells, cytoplasm homogeneity and integrity and were divided into two groups: G1 – considered morphologically healthy (oocytes with compact cumulus and more than three cell layers, intact cytoplasm, evenly granular and homogenous coloration); and G2 – considered morphologically less viable or with some degree of atresia (oocytes that presented less than three layers of cumulus cells and/or expanded cumulus, with cumulus granular appearance, heterogeneous or dense and/or shrunken cytoplasm).
In vitro maturation (IVM)
In vitro maturation of the oocytes was carried out in follicular fluid as described previously (Caillaud et al., Reference Caillaud, Dell'Aquila, De, Nicassio, Lacalandra, Goudet and Gerard2008). Briefly, equine follicular fluid was collected from follicles smaller than 30 mm, centrifuged at 14,000 rpm for 10 min, filtered through a 22 μm filter and heat-inactivated at 56°C for 30 min. For IVM, the COC were incubated for 36 h at 38.7°C and 5% CO2. At the end of the incubation period the COC were denuded mechanically by repeated pipetting in a solution of 80 IU/ml of type-I hyaluronidase (Sigma-Aldrich). Groups of 60 oocytes and the corresponding cumulus cells were cryopreserved in 50 μl of TRIzol Reagent (Invitrogen) for RNA extraction and subsequent assessment of rates of gene expression by real-time reverse transcription PCR (qRT-PCR). As controls of the IVM process, the COC were classified into G1 and G2, denuded and total RNA was extracted immediately after collection. Nine hundred and sixty COC were divided among the experimental groups, four replicates were performed on pools of 60 COC.
To determine the stage of nuclear maturation of the oocytes upon collection and after IVM, 10% were assessed by staining. The maturation rate was determined by Hoechst 33342 staining (Sigma-Aldrich using a dye solution (10 μg/ml) and denuded oocytes were incubated for 10 min at 38.7°C. Slides were evaluated using an epifluorescence microscope (Olympus), with filter wavelength of BP330–385 nm. Immature oocytes were those oocytes presenting a germinal vesicle (GV) with a single condensed mass associated with the nucleolus; or germinal vesicle breakdown (GVBD) with an irregular envelope surrounding disperse condensed chromatin. Metaphase I (MI) oocytes were those presenting the first metaphase plate. In oocytes at MII the metaphase plate was located peripherally in the ooplasm and polar body in the perivitelline space. Oocytes with abnormal chromatin configurations or no chromatin visible were considered to be degenerating.
Total RNA isolation, reverse transcription and real-time PCR (qRT-PCR)
Total RNA was extracted from the COC stored in TRIzol as described by the manufacturer. The extracted RNA was quantified using the Qubit Fluorometer (Invitrogen), with the Quant-iT RNA BR Assay Kit following the manufacturer's instructions and standardized to a concentration of 4 ng/ml (equivalent to 60 oocytes). cDNA was produced using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer's instructions. The cDNA was then used as template for the qRT-PCR, Stratagene Mx3005P Real-Time PCR System (Agilent Technologies), reaction using the Platinum Sybr Green Kit (Invitrogen). The primer pairs for the qRT-PCR were designed using Vector NTI 11 software (Invitrogen, USA) using the sequences for each of the target genes, Table 1. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (Smits et al., Reference Smits, Goossens, Van, Govaere, Hoogewijs, Vanhaesebrouck, Galli, Colleoni, Vandesompele and Peelman2009) was used for normalization of the target gene expression data.
Primers used in qRT-PCR for equine oocytes
Data processing and statistical analysis
Data processing and statistical analysis were performed using the software REST 2009. The rate of gene expression was calculated relative to the expression of the GAPDH gene, based on the method of Pfaffl and colleagues (Pfaffl et al., Reference Pfaffl, Horgan and Dempfle2002). The various groups were analysed using integrated randomization and bootstrapping methods to compare expression ratios, P-values ≤ 0.05 were considered to be significant.
Results
Differentiation and COC in vitro maturation
Some 1160 COC were recovered from 528 ovaries. Immediately after collection, 76% (76/100) of oocytes were at the germinal vesicle stage, 17% (17/100) were at the germinal vesicle breakdown stage and 7% (7/100) were at the degenerated stage. The COC were divided into two groups: G1 – considered morphologically healthy; and G2 – considered morphologically less viable or with some degree of atresia, see Fig. 1. The average rate of IVM was 51%, with a rate of 54% (27/50) for the G1 group and 48% (24/50) for the G2 group.
(A) Oocytes classified as healthy morphological group, G1. (B) Oocyte classified as morphological group with some signs of degeneration, G2.
Relative expression of the Bax, Bcl-2, survivin and p53 genes
Overall our results showed that survivin gene expression was significantly downregulated while p53 expression was significantly upregulated in cumulus cells in comparison with COC. Furthermore, p53 expression was higher in both immature and in vitro matured cumulus cells.
The survivin gene showed higher expression in in vitro matured oocytes from morphological group G1 than in cumulus cells from these oocytes (P < 0.02). The p53 gene showed a lower expression in G1 in vitro matured oocytes compared to G1 in vitro matured cumulus cells (P = 0.007). However, the same relation was not observed between mature oocytes and cumulus cells mature G2. Figure 2 shows the data on survivin gene expression and the data on p53 gene expression in oocyte and cumulus cells of G1 and G2 groups. When the expression was compared between oocytes and cumulus cells according to morphology and maturation, differences for genes Bcl-2 and Bax (P > 0.05) were not detected.
Normalized CT values of the mRNA levels of the four apoptotic genes, Bcl-2, Bax, survivin and p53. (A) Immature oocytes and cumulus cells from groups G1 and G2. (B) In vitro matured oocytes and cumulus cells from groups G1 and G2. Significant differences are indicated *P < 0.05, **P < 0.01 and ***P < 0.001. iCum: immature cumulus cells; iOoc: immature oocytes; mCum: matured cumulus cells; mOoc: matured oocytes.
In this study, no difference was detected in the expression of apoptotic genes analysed in immature oocytes and after IVM (P > 0.05). In oocytes from different morphological groups, G1 and G2, there was no difference in Bcl-2, Bax, survivin and p53 gene expression in immature and in vitro maturated oocytes. Among the groups of oocytes according to morphological classification, G1 and G2, there was no difference in Bcl-2, Bax, survivin and p53 gene expression (P > 0.05).
Changes in gene expression of Bcl-2, survivin and p53 (P > 0.05) were not observed among cumulus cells. Between the morphological classification groups G1 and G2 there was observed difference in the expression of cumulus cells for the pro-apoptotic Bax gene. The expression of Bax was higher in cells of the morphological group G1 than G2 (P = 0.02) (Fig. 2).
Discussion
This study evaluated the expression of Bax, Bcl-2, survivin and p53 genes during IVM of equine oocytes, to compare expression between different morphological parameters of oocytes and cumulus cells from these complexes. Gene expression studies in equine species are relatively scarce (Smits et al., Reference Smits, Goossens, Van, Govaere, Hoogewijs, Vanhaesebrouck, Galli, Colleoni, Vandesompele and Peelman2009), this was the first report of gene expression in equine COC, allowing us to elucidate the pattern of expression of apoptotic genes.
We observed different expression of anti-apoptotic gene survivin and pro-apoptotic gene p53 between oocytes and cumulus cells. Equine morphologically viable oocytes expressed a higher rate of survivin and lower rate of p53 than cumulus cells. Gene expression in cumulus cells, including genes involved in the apoptotic process, provides evidence that embryo viability is reflected in differential gene expression in the cumulus cells (van Montfoort et al., Reference van Montfoort, Geraedts, Dumoulin, Stassen, Evers and Ayoubi2008). The bidirectional communication between the oocyte and companion somatic cells is essential for development of an egg competent to undergo fertilization and embryogenesis (Matzuk et al., Reference Matzuk, Burns, Viveiros and Eppig2002). An increase in apoptotic cumulus cells has been related to a decrease of mature oocytes and to a decreased ability to be fertilized in stimulated IVF cycles (Host et al., Reference Host, Mikkelsen, Lindenberg and Smidt-Jensen2000). Our results indicated that the survivin and p53 proteins synthesized act on survival and early development of equine embryos.
Upon resumption of meiosis GVBD, oocytes become transcriptionally quiescent and must rely on pools of RNA accumulated during the growth phase for protein synthesis (Wassarman & Letourneau, Reference Wassarman and Letourneau1976). In a study of oocytes and cumulus cells of bovine, ovine, porcine, canine, feline and murine, it was demonstrated that oocyte RNA features were repeatable whether maturation occurred in vitro or in vivo, and were similar between the phases of nuclear maturation of the germinal vesicle and MII oocytes (Payton et al., Reference Payton, Rispoli and Edwards2010). The differences in features of total RNA from oocytes versus cumulus was reported, oocytes are contained within growing antral follicles which are in the large part transcriptionally inactive while cumulus cells are transcriptionally active. Furthermore, oocytes contain maternal pools of RNA, protein and energy stores that accumulated during the growth phase, a period during which features of oocyte RNA are more like somatic cells (Payton et al., Reference Payton, Rispoli and Edwards2010).
Survivin expression was appointed as a possible marker for good quality in vitro developmental capacity of bovine follicular oocytes (Jeon et al., Reference Jeon, Kim, Tae, Lee, Lee, Kim, Jeong, Cho, Kim, Lee, Riu, Cho and Park2008). The expression of survivin was related to the quality of COCs, their developmental competence and the quality of in vitro produced blastocysts in bovine (Jeon et al., Reference Jeon, Kim, Tae, Lee, Lee, Kim, Jeong, Cho, Kim, Lee, Riu, Cho and Park2008). Survivin protein is known as a bifunctional protein that suppresses apoptosis and regulates cell division. Survivin acts as an inhibitor of apoptosis by linking directly to the caspases, it has been shown to inhibit the caspase 3 activity directly, preventing the formation of apoptosome (Kawamura et al., Reference Kawamura, Sato, Fukuda, Kodama, Kumagai, Tanikawa, Nakamura, Honda, Sato and Tanaka2003; Altieri, Reference Altieri2010).
The tumor suppressor gene p53 is an important mediator in response to cellular stress (Dhali et al., Reference Dhali, Anchamparuthy, Butler, Pearson, Mullarky and Gwazdauskas2007). The transcriptional activity of p53 is required for cell death in some systems, its nuclear translocation is required for transcription of the gene Bax (Sabbatini et al., Reference Sabbatini, Lin, Levine and White1995). However, p53 gene expression appears to have no correlation with morphological quality of bovine embryos (Melka et al., Reference Melka, Rings, Holker, Tholen, Havlicek, Besenfelder, Schellander and Tesfaye2009). A significantly higher expression of p53 from oocyte to four-cell stage as compared with that of the later pre-implantation stages was reported, suggesting p53-independent apoptosis in bovine embryos (Melka et al., Reference Melka, Rings, Holker, Tholen, Havlicek, Besenfelder, Schellander and Tesfaye2009). Equine oocytes are characterized by a large amount of lipid droplets in their cytoplasm, in association with the mitochondrion and smooth endoplasmic reticulum (Tremoleda et al., Reference Tremoleda, Stout, Lagutina, Lazzari, Bevers, Colenbrander and Galli2003; Ambruosi et al., Reference Ambruosi, Lacalandra, Iorga, De, Mugnier, Matarrese, Goudet and Dell'Aquila2009), suggesting that the oocytes and embryos of this species are more sensitive to oxidative stress and apoptosis (Ambruosi et al., Reference Ambruosi, Lacalandra, Iorga, De, Mugnier, Matarrese, Goudet and Dell'Aquila2009). IVM of oocytes is an essential step for in vitro production of embryos, however the conditions under which the oocytes are subjected may result in changes in gene expression in these cells, as a response to injuries suffered.
Expression of the Bax gene has been identified in oocytes, granulosa cells and luteal cells of various species and levels of Bax expression appear to be positively correlated with apoptosis in each of these lineages. Bax and p53 upregulation has been associated with apoptosis in granulosa cells (Zwain & Amato, Reference Zwain and Amato2001). In the morphological classification groups G1 and G2 difference in the expression of cumulus cells for the gene pro-apoptotic Bax was observed, the expression of Bax is higher in cells of the morphological group G1, and Bcl-2 has not changed. However, in the study by Filali et al. (Reference Filali, Frydman, Belot, Hesters, Gaudin, Tachdjian, Emilie, Frydman and Machelon2009) Bcl-2 mRNA expression was found to be significantly higher in cumulus cells associated with mature oocytes than those associated with immature oocytes, whereas Bax mRNA concentrations did not vary in cumulus cells from either source.
In this study, no difference was detected in the expression of apoptotic genes analysed in immature oocytes and after the IVM period, and in oocytes from different morphological groups, G1 and G2. There was no difference in gene expression of Bcl-2, Bax, survivin and p53 in immature oocytes and in in vitro maturated oocytes. Similar results were published comparing the expression levels of anti-apoptotic gene Bcl-2 in different morphological groups of bovine oocytes in vivo matured, and found no difference in the quantification of transcripts to this gene (Pretheeban et al., Reference Pretheeban, Gordon, Singh, Perera and Rajamahendran2009). The potential development of in vitro produced embryos depends mainly on the quality of oocytes from which they originate, with the selection of oocytes based primarily on morphological features. While, Yang & Rajamahendran (Reference Yang and Rajamahendran2002) reported higher levels of expression of the anti-apoptotic gene Bcl-2 in oocytes considered morphologically healthy, Li et al. (Reference Li, Liu, Cang, Wang, Jin, Ma and Shorgan2009) observed initial characteristics of apoptosis in immature COC, which was confirmed by qRT-PCR transcripts of Bax and Bcl-2 showing that the dynamic change in the transcriptional profile of Bax corresponded with the occurrence of apoptosis and early development of oocytes, while the pattern of Bcl-2 transcription showed a contrasting pattern.
In conclusion it was observed that equine morphologically viable oocytes expressed a greater rate of survivin and lower rate of p53 than cumulus cells. No difference was detected in the expression of apoptotic genes analysed in immature oocytes compared with after the IVM period, or in oocytes from different morphological groups, but higher expression of the pro-apoptotic gene Bax in G1 compared with the G2 group was observed in cumulus cells. In conclusion, other genes involved in cell survival and viability need to be studied, higher number of oocytes, corresponding to different stages of meiotic maturation, are targets for our subsequent studies. We believe that our data can contribute to identification of gene regulation during maturation and increase the knowledge of biology of the oocyte. It signals the progress in understanding the molecular events involved in communication and IVM of the COC, indicating possible markers of viability and competence.
Acknowledgements
This work was supported by grants from CNPq and FAPERGS, Brazil. P.M.M.L, V.F.C., C.K. and K.B. were supported by scholarships from CAPES, Brazil and J.C.D. and O.A.D. received a research fellowship from CNPq.
