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Cellular and molecular alterations of buffalo oocytes cultured under two different levels of oxygen tension during in vitro maturation

Published online by Cambridge University Press:  24 February 2021

Nasser Ghanem Open the ORCID record for Nasser Ghanem [Opens in a new window] ,
Dalia Abd-El Rahman Ahmed ,
Sherif Mohamed Dessouki ,
Marwa Said Faheem ,
Ahmed Yehia Gad ,
Jaana Peippo  and
Ashraf Hesham Barkawi
Nasser Ghanem*
Affiliation:
Department of Animal Production, Faculty of Agriculture, Cairo University, Giza, Egypt Cairo University Research Park, Faculty of Agriculture, Cairo University, Giza, Egypt
Dalia Abd-El Rahman Ahmed
Affiliation:
Department of Animal Production, Faculty of Agriculture, Cairo University, Giza, Egypt
Sherif Mohamed Dessouki
Affiliation:
Department of Animal Production, Faculty of Agriculture, Cairo University, Giza, Egypt
Marwa Said Faheem
Affiliation:
Department of Animal Production, Faculty of Agriculture, Cairo University, Giza, Egypt
Ahmed Yehia Gad
Affiliation:
Department of Animal Production, Faculty of Agriculture, Cairo University, Giza, Egypt
Jaana Peippo
Affiliation:
Natural Resources Institute Finland (Luke), Myllytie 1, FI-31600 Jokioinen, Finland
Ashraf Hesham Barkawi*
Affiliation:
Department of Animal Production, Faculty of Agriculture, Cairo University, Giza, Egypt
*
Authors for correspondence: Ashraf Hesham Barkawi. Cairo University, Faculty of Agriculture, Department of Animal Production, El-Gamaa Street, 12613 Giza, Egypt. E-mail: ashrafb53@yahoo.com; Nasser Ghanem. Cairo University, Faculty of Agriculture, Department of Animal Production, El-Gamaa street, 12613 Giza, Egypt. E-mail: nassergo@agr.cu.edu.eg
Authors for correspondence: Ashraf Hesham Barkawi. Cairo University, Faculty of Agriculture, Department of Animal Production, El-Gamaa Street, 12613 Giza, Egypt. E-mail: ashrafb53@yahoo.com; Nasser Ghanem. Cairo University, Faculty of Agriculture, Department of Animal Production, El-Gamaa street, 12613 Giza, Egypt. E-mail: nassergo@agr.cu.edu.eg
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Summary

This study was conducted to monitor the cellular and molecular changes of buffalo cumulus–oocytes complexes (COCs) cultured under high or low oxygen levels. Morphologically good quality COCs (n = 1627) were screened using brilliant cresyl blue (BCB) staining and placed into three groups (BCB+, BCB− and control). All groups of COCs were cultured under low (5%) or high (20%) oxygen tensions. Intracellular and molecular changes including oocyte ultrastructure, lipid contents, mitochondrial activity and transcript abundance of genes regulating different pathways were analyzed in the matured oocyte groups. The results revealed that oxygen tension did not affect cumulus expansion rates, however the BCB+ group had a higher (P ≤ 0.05) expansion rate compared with the BCB− group. BCB− oocytes recorded the lowest meiotic progression rate (P ≤ 0.05) under high oxygen levels that was linked with an increased level of reactive oxygen species (ROS) compared with the BCB+ oocytes. Ultrastructure examination indicated that BCB+ oocytes had a higher rate of cortical granules migration compared with BCB− under low oxygen tension. In parallel, our results indicated the upregulation of NFE2L2 in groups of oocytes cultured under high oxygen tension that was coupled with reduced mitochondrial activity. In contrast, the expression levels of MAPK14 and CPT2 genes were increased (P ≤ 0.05) in groups of oocytes cultured under low compared with high oxygen tension that was subsequently associated with increased mitochondrial activity. In conclusion, data from the present investigation indicated that low oxygen tension is a favourable condition for maintaining the mitochondrial activity required for nuclear maturation of buffalo oocytes. However, low-quality oocytes (BCB−) responded negatively to high oxygen tension by reducing the expression of gene-regulating metabolic activity (CPT2). This action was an attempt by BCB− oocytes to reduce the increased levels of endogenously produced ROS that was coupled with decreased expression of the gene controlling meiotic progression (MAPK14) in addition to nuclear maturation rate.

Keywords

BCB oocytesGene expressionOxidative stressUltrastructure

Information

Type
Research Article
Information
Zygote , Volume 29 , Issue 4 , August 2021 , pp. 314 - 324
Copyright
© The Author(s), 2021. Published by Cambridge University Press

Introduction

Buffaloes play a pivotal role in the Egyptian agricultural economy. This is attributed to their substantial contribution to meat and milk production. However, buffaloes reproduce at a very low rate due to silent ovulation, unordinary follicular dynamics, and low numbers of ovarian follicles (Barkawi et al., Reference Barkawi, Hafez, Ibrahim, Ashour, El-Asheeri and Ghanem2008). Therefore, assisted reproductive technologies (ARTs) have become an essential tool to enhance buffalo reproductive efficiency (Madan et al., Reference Madan, Das and Palta1996). The efficiency of in vitro embryo production is extremely important for producing viable embryos (Feugang et al., Reference Feugang, Camargo-Rodriguez and Memili2009). Despite the increased rates of in vitro embryo production (IVP) over the last decades, the quality of IVP embryos is still lower than that of their in vivo counterparts. This is mainly attributed to the composition of the culture medium, its pH, and the choice of oxygen tension used during IVP (Du Plessis et al., Reference Du Plessis, Makker, Desia and Agrawal2008).

Suboptimal conditions of in vitro culture ultimately compromise blastocyst quality due to increased accumulation of reactive oxygen species (ROS) (Oyamada and Fukui, Reference Oyamada and Fukui2004). In fact, ROS could be increased by high oxygen levels during any step of IVP (Goto et al., Reference Goto, Noda, Mori and Nakano1993; Bavister, Reference Bavister1995; Karagenc et al., Reference Karagenc, Sertkaya, Ciray, Ulug and Bahçe2004). The normal physiological concentration of oxygen in the reproductive tract of bovine females is approximately 5% (Bavister, Reference Bavister1995). High levels of oxygen (20%) are responsible for inducing oxidative stress during in vitro preimplantation development (Karagenc et al., Reference Karagenc, Sertkaya, Ciray, Ulug and Bahçe2004). Ovarian follicles are dynamic structures that undergo growth, ovulation, and luteinization processes during the reproductive cycle. Due to the lack of a direct blood supply, oxygen concentration decreases in follicular fluid during follicular maturation, reaching its lowest level in preovulatory follicles (Fischer et al., Reference Fischer, Künzel, Kleinstein and Gips1992). The dissolved oxygen in human ovarian follicular fluid is predicted to be 1.5–6.7% based on mathematical modelling (Redding et al., Reference Redding, Bronlund and Hart2008). Several studies have confirmed these values and the estimated level of dissolved oxygen ranges between 1% and 5% (Van Blerkom et al., Reference Van Blerkom, Antczak and Schrader1997; Huey et al., Reference Huey, Abuhamad, Barroso, Hsu, Kolm, Mayer and Oehninger1999),

There is an equilibration system that keeps the generation of oxygen species in balance in any living cell (Whitaker, Reference Whitaker2007). When this system is compromised and the available antioxidants cannot maintain oxygen concentrations within the appropriate level, an overabundance of ROS is generated and induced oxidative stress leads to programmed cell death (Fiers et al., Reference Fiers, Beyaert, Declercq and Vandenabeele1999). Various types of ROS, such as superoxide (O2−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH) can trigger oxidative stress. These types of ROS originate from the environment, as well as from within oocytes and embryos as a by-product of energy metabolism, in particular from mitochondrial phosphorylation and glycolysis (Guerin et al., Reference Guerin, El Mouatassim and Menezo2001; Balaban et al., Reference Balaban, Nemoto and Finkel2005).

Oxidative stress has broad effects on the reproductive system and has resulted in impairment of the functionality of ovarian steroidogenesis, ovulation (Agarwal et al., Reference Agarwal, Gupta and Sharma2005), embryogenesis (Bedaiwy et al., Reference Bedaiwy, Falcone, Mohamed, Aleem, Sharma, Worley, Thornton and Agarwal2004), and maintenance of pregnancy (Myatt and Cui, Reference Myatt and Cui2004). It is noteworthy that oxidative stress has negatively affected the nuclear maturation of oocytes (Cetica et al., Reference Cetica, Pintos, Dalvit and Beconi2001; Ali et al., Reference Ali, Biloudeau and Sirard2003). Accumulation of ROS increases the incidence of chromosomal errors, disorganization of the microtubule, disassembly of meiotic spindles, and finally has compromised the quality of mice oocytes during IVM (Tarin et al., Reference Tarin, Vendrell, Ten, Blanes, van Blerkom and Cano1996; Choi et al., Reference Choi, Banerjee, Falcone, Bena, Agarwal and Sharma2007; Tamura et al., Reference Tamura, Takasaki, Miwa, Taniguchi, Maekawa, Asada, Taketani, Matsuoka, Yamagata, Shimamura, Morioka, Ishikawa, Reiter and Sugino2008). Furthermore, oxidative stress has a harmful effect on subsequent preimplantation development by perturbation of various cellular organelles such as mitochondria in addition to biochemical composition such as lipids (Halliwell, Reference Halliwell1989; Nasr-Esfahani et al., Reference Nasr-Esfahani, Aitken and Johnson1990). Buffalo oocytes have a high lipid content, which makes them less resistant to oxidative stress caused by ROS during in vitro maturation (Boni et al., Reference Boni, Santella, Dale, Roviello, Palo and Barbieri1992).

Therefore, the current study was designed to determine the cellular and molecular changes found in buffalo oocytes selected by BCB staining (good quality vs. bad quality) and matured in vitro under two levels of oxygen (20% vs. 5%).

Materials and methods

All chemicals were purchased from Sigma-Aldrich unless otherwise indicated.

Experimental design

Cumulus–oocyte complexes (COCs) were distributed into three groups that were divided into two sub-groups based on oxygen tension, as follows: control (C5% n = 224, C20% n = 348); BCB+ (BCB+ 5% n = 269, BCB+ 20% n = 358) and BCB− (BCB− 5% n = 181 BCB− 20% n = 247). Different parameters were evaluated for oocytes following IVM under these two conditions.

In vitro assessment of oocytes maturation

Buffalo ovaries from cyclic animals were collected from a local Egyptian slaughterhouse and kept at 34–37°C in a thermos container in 0.9% NaC1 supplemented with 50 µg/ml gentamycin. COCs were aspirated from 2–8 mm follicles using an 18-gauge needle attached to a 10-ml disposable syringe. The aspirated COCs were classified morphologically under a stereomicroscope based on the number of cumulus layers and ooplasm homogeneity (Faheem and Barkawi, Reference Faheem and Barkawi2012). After staining with BCB, COCs were washed twice with modified Dulbecco’s phosphate-buffered saline (mDPBS). The stained COCs were examined under a stereomicroscope and divided into two groups based on their cytoplasm coloration; oocytes with any degree of blue coloration in the cytoplasm were considered BCB+, while oocytes without blue cytoplasm were considered BCB− (Manjunatha et al., Reference Manjunatha, Gupta, Devaraj, Ravindra and Nandi2007). The control group was not exposed to BCB stain. All groups of COCs were washed twice in the maturation medium (Faheem et al., Reference Faheem, Baron, Carvalhais, Chaveiro, Pavani and Moreira da Silva2015), which consisted of TCM-199 supplemented with 10% FBS, 5 µg/ml FSH, 1 µg/ml estradiol-17β, 0.15 mg/ml glutamine, 22 µg/ml Na-pyruvate and 50 µg/ml gentamycin. In total, 10–15 COCs from each group were seeded into drops of 100 µl maturation medium under mineral oil and incubated at 38.5°C in 5% CO2, 90% N2 and 5% O2 (triple gas mix); or 5% CO2 + N2 75% and 20% O2, respectively. The phase of oocytes nuclear maturation was assessed after 22–24 h of IVM. Oocytes were denuded, then fixed in a Petri dish containing fixation solution (methanol:glacial acetic acid, 3:1) and left for 24 h. After the fixation period, oocytes were placed on glass slides and stained with 1% aceto orcein for 5 min, then covered with a glass coverslip before examination under an inverted microscope. Matured oocytes were evaluated in relation to their meiotic stage as: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), or metaphase II (MII) stages (Faheem and Barkawi, Reference Faheem and Barkawi2012).

Preparation of mature oocytes for transmission electron microscopy (TEM)

After 22–24 h of IVM, the cumulus cells were removed by repeated pipetting of intact COCs into IVM medium supplemented with 0.25% of hyaluronidase. Denuded oocytes were fixed in 2.5% glutaraldehyde supplemented with 0.1 M sodium phosphate buffer (pH 7.4) at room temperature for 15 min, then washed three times in sodium phosphate buffer for 30 min each. Next, oocytes were treated with osmium tetroxide (OsO4 1%) and dehydrated in increasing concentrations of ethanol before treatment with 100% ethanol twice for 15 min each, according to Anguish and Coonrod (Reference Anguish and Coonrod2013). Dehydrated oocytes were infiltrated with Spurr’s epoxy resin, and then were sectioned (90 µm thick) using an ultramicrotome (Leica model EM-UC6, Japan) and finally mounted on copper grids (400 mesh). Stained sections were examined under a JEOL transmission electron microscope (JEM-1400 TEM, Japan). Images of mitochondria, lipid droplets, and cortical granules, were captured to evaluate their cellular structure. Total mitochondrial number per μm2 was counted precisely (n = 6 oocytes × 4 fields/oocyte). The number of each type of peripheral and central mitochondrial distribution was counted per μm2. In addition, the number and distribution of lipid droplets per μm2 were determined (n = 6 oocytes × 2 fields/oocyte). Finally, the cortical granule migration pattern was calculated.

Evaluation of oocyte cytoplasmic lipid content

Nile red stock solution was prepared by dissolving 1 mg/ml of the stain in DMSO and then stored in the dark. Briefly, matured oocytes (n = 10/group) were denuded from cumulus cells and washed twice in mDPBS. The washed oocytes were fixed in 4% paraformaldehyde in PVP-PBS for 30 min. The fixed oocytes were first stained in 500 ml of a 10 mg/ml Nile red solution adjusted in PVP-PBS solution (0.3% [wt/vol] PVP in 1 M PBS) for 3 h. Stained oocytes were washed three times in PVP-PBS (15 min each) as detailed in Ghanem et al. (Reference Ghanem, Ha, Fakruzzaman, Bang, Lee and Kong2014).

Evaluation of oocyte mitochondrial activity

Mitochondrial activity was measured using a commercial kit (MitoTracker Green FM; Invitrogen, Carlsbad, CA, USA) based on Ghanem et al. (Reference Ghanem, Ha, Fakruzzaman, Bang, Lee and Kong2014). Oocytes stained with Nile red were subsequently used for mitochondrial activity measurement. Oocytes were washed three times in 500 µl PVP-PBS (15 min each) and incubated for 5 min with 125 nM of MitoTracker dissolved in PVP-PBS. The stained oocytes were washed twice in PVP-PBS (10 min each). Next, oocytes were mounted in glycerol on clean slides. Finally, a coverslip was placed over the oocytes (without compressing). An epifluorescence microscope was used to excite both Nile red and MitoTracker Green FM lipophilic fluorescent dyes (580–596 nm).

Measurement of ROS

For quantification of H2O2, matured oocytes were incubated in PVP-PBS supplemented with 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCHFDA) for 20 min at 38.5°C (Fakruzzaman et al., Reference Fakruzzaman, Ghanem, Bang, Ha, Lee, Sohn, Wang, Lee and Kong2015). After incubation, oocytes were washed three times with PVP-PBS, then mounted onto glass slides, and visualized immediately under an epifluorescence microscope at 490 nm excitation and 525 nm emission. Images analysis was carried out using ImageJ software (https://imagej.nih.gov/ij/download.html).

Gene expression

RNA extraction and cDNA synthesis

Three replicates from each experimental group were used for RNA isolation and cDNA synthesis (n = 15–20 matured oocytes/replicate). Following the manufacturer’s instructions, total RNA was extracted using the PicoPure RNA isolation kit (MDS Analytical Technologies GmbH, Ismaning, Germany). Matured oocytes were mixed thoroughly with lysis buffer and incubated at 42°C for 30 min. The complete lysate was loaded into a pre-conditioned spin column and centrifuged shortly for 2 min at 3600 g to allow the RNA to bind to the spin column. DNA was removed using an on-column RNase-free DNase kit (Qiagen GmbH, Hilden, Germany). The column was washed with two different washing buffers. Finally, RNA was eluted with 12 µl RNase-free water.

For each sample, cDNA synthesis was performed using a high-capacity cDNA reverse transcription kit (Life Technologies Corporation, California, USA). Total RNA from all samples was adjusted by adding RNase-free water to achieve the same concentration of RNA. A 14.2 μ1 volume of RNA sample was added to 1 μ1 10× RT buffer, 0.8 μ1 25× dNTP, 2 μ1 random primers, 1 μ1 RNase inhibitor, and 1 μ1 Multiscribe reverse transcriptase. The mixture was incubated in a cycle of 25°C for 10 min, 37°C for 120 min, 85°C for 5 min, and holding at 4°C. The cDNA samples were stored at −20°C until real-time polymerase chain reaction (PCR) quantification.

Real-time PCR analysis

Real-time PCR primers (Table 1) were designed using Primer3 software (http://primer3.wi.mit.edu//). GAPDH was used as a reference gene for real-time PCR quantification. The PCR mixture was composed of 10 μl Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, California, USA), 0.2 μl forward primer, 0.2 μl reverse primer, 7.6 μl nuclease-free water, and 2 μl cDNA. Thermal cycling conditions were set as 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 30 s and a final step at 60°C for 1 min. After the end of the last cycle, a dissociation curve was generated by starting the fluorescence acquisition at 60°C and taking measurements at 7-s intervals until the temperature reached 95°C. Relative expression was calculated using the relative quantification method (2-ΔΔCT method) (Bermejo-Alvarez et al., Reference Bermejo-Alvarez, Rizos, Rath, Lonergan and Gutiérrez-Adan2010).

Table 1.

List of primers used for quantitative real-time PCR analysis

Statistical analysis

All data were analyzed using analysis of variance (ANOVA) and the general linear model procedure (SAS, 2004), followed by multiple pairwise comparisons using Tukey’s test. The data were expressed as mean ± standard error. The effect of oxygen level (5% vs. 20%) and oocyte type (BCB+ vs. BCB− vs. control) and the interaction between these two factors were tested by applying the following model:

$${{\rm{Y}}_{{\rm{ijkl}}}} = {\rm{\mu }} + {{\rm{R}}_{\rm{i}}} + {{\rm{A}}_{\rm{j}}} + {({{\rm{R}}_{\rm{i}}}*{{\rm{A}}_{\rm{j}}})_{{\rm{ijk}}}} + {{\rm{e}}_{{\rm{ijkl}}}}$$

where: Y ijk1 = individual observation; µ = overall mean; Ri = effect of BCB staining (BCB+ = 1 and BCB− = 2); Aj = effect of oxidative stress (oxygen level) (5% = 1 and 20% = 2); (Ri*Aj)ijk = the interaction between BCB and oxidative stress; eijk1 = experimental error supposed to be randomly distributed (0, σ2). A probability value (P-value) ≤ 5% was considered to be statistically significant.

Results

In vitro maturation assessment

The nuclear maturation of buffalo COCs was assessed to gain insight into the possible interaction of different levels of oxygen tension on the first crucial step of oocyte developmental competence when classified into different qualities using BCB staining. Oxygen tension had no significant effect on cumulus expansion rates (Table 2), however BCB+ had a higher (P ≤ 0.05) expansion rate (95.49%) than BCB− (89.73%). Conversely, oxygen tension had a significant effect on nuclear maturation rate (oocytes that reached the MII stage). Regardless of oocyte quality, high oxygen level (20%) reduced (P ≤ 0.05) the rate of oocyte maturation by about 20% (Table 2). Moreover, BCB− oocytes had a lower (P ≤ 0.05) rate of MII stage under the high oxygen level (20%) compared with their counterparts cultured under low oxygen tension (Fig. 1).

Table 2.

Cumulus expansion and nuclear maturation rate of buffalo oocytes cultured under 5% and 20% oxygen tensions during in vitro maturation

Abbreviations: bp, base pair; PCR, polymerase chain reaction.

Values with different superscripts (a, b) within the same row and experimental category (oxygen level, quality) indicate significant difference at P ≤ 0.05.

Figure 1.

Cumulus expansion and nuclear maturation rate of buffalo oocytes cultured under 5% and 20% oxygen tension during in vitro maturation. Bars with different superscripts (a–c) indicate significant difference at P ≤ 0.05. Cumulus expansion rate: numbers of oocyte that have completely or partially expanded in cumulus layers/total numbers of cultured oocytes. Nuclear maturation rate (%): oocytes that have reached telophase and MII stages.

Cortical granules migration

The migration of cortical granules is considered as a marker of cytoplasmic maturation of COCs. Therefore, we examined the distribution of these cellular organelles in oocytes of different qualities and after in vitro maturation under the two oxygen levels. The total number of cortical granules (Table 3) was affected by the oxygen level, being significantly higher for oocytes matured under 5% (4.72 ± 0.11) than 20% (3.33 ± 0.11). Similarly, the number of migrated (peripheral distributed) cortical granules was increased (P ≤ 0.05) under low (3.55 ± 0.26) compared with high (2.44 ± 0.26) oxygen tension. In addition, BCB+ and BCB− oocytes had higher cortical granule numbers compared with the control group. The number of migrated cortical granules was higher in BCB+ compared with the BCB− and control groups under low oxygen level (Fig. 2), however there were no differences among oocytes groups under high oxygen level. Moreover, there were no differences among all experimental groups concerning non-migrated cortical granules under high or low oxygen tensions. In general, oocytes cultured under high oxygen tension had lower (P ≤ 0.05) cortical granule numbers compared with those cultured under low oxygen tension (Table 3 and Fig. 3).

Table 3.

Cortical granules distribution (migrated and non-migrated) and numbers in vitro matured buffalo oocytes cultured under low (5%) or high oxygen tensions (20%)

Values with different superscripts (a, b) within the same row and experimental category (oxygen level, quality) indicate significant difference at P ≤ 0.05.

Figure 2.

Percentage of cortical granules distribution (migrated and non-migrated) in buffalo matured oocytes cultured under low (5%) or high oxygen tension (20%) during in vitro maturation. Bars with different superscripts (a–c) indicate significant difference at P ≤ 0.05. Migrated: moved peripherally beneath the plasma membrane. Non-migrated: diffused in the cytoplasm.

Figure 3.

Representative photographs of the cortical granules of in vitro matured Egyptian buffalo oocytes taken with a transmission electron microscope (×6000).

Lipid droplet number, distribution, and fluorescence intensity

Integration of the data on number and distribution of cellular organelles such as mitochondria with their activity measured by fluorescence intensity to understand their possible role during in vitro maturation of COCs cultured under two different levels of oxygen tension has not been described previously. As shown in Fig. 4, the intracellular lipid droplet number was increased in oocytes cultured under low compared with high oxygen tension. However, there were no significant differences in this parameter among control, BCB+, and BCB− groups.

Figure 4.

Percentage of lipid droplet distribution (peripheral and central) of buffalo mature oocyte cultured under low (5%) or high oxygen tensions (20%) during in vitro maturation. Bars with different superscripts (a–c) indicate significant difference at P ≤ 0.05. Central: located in the centre of the cytoplasm. Peripheral: moved peripherally beneath the plasma membrane.

The numbers of central intracellular lipid droplets were similar under the two oxygen levels and in all oocyte quality groups. However, the lowest number of peripheral lipid droplets was recorded in oocytes cultured under low oxygen tension and BCB− oocytes. Intracellular lipid fluorescence intensity was higher (P ≤ 0.05) in the control and BCB− oocytes compared with BCB+ oocytes cultured under low oxygen tension. In addition, there were no differences in lipid fluorescence intensity among all groups of oocytes cultured under high oxygen tension (Fig. 5).

Figure 5.

Fluorescence intensity of intracellular lipid content per buffalo mature oocytes cultured under low (5%) or high oxygen tensions (20%) during in vitro maturation. Bars with different superscripts (a–c) indicate significant difference at P ≤ 0.05.

Mitochondrial number, distribution, and fluorescence intensity

The total mitochondrial number was similar in all oocyte groups regardless of oxygen level and quality of oocytes (Figs 6 and 7). Moreover, the central mitochondrial distribution was more intense than the peripheral type in all oocyte groups under the two oxygen levels (Table 4).

Figure 6.

Percentage of mitochondria distribution (peripheral and central) of mature buffalo oocytes cultured under low (5%) or high oxygen tensions (20%) during in vitro maturation. Central: diffused in cytoplasm. Peripheral: moved peripherally beneath the plasma membrane.

Figure 7.

Electron micrograph of Egyptian buffalo oocytes showing oval (OM), round (RM), vacuolated (VM), distorted mitochondrial membrane (DMM) and deformed (DM) forms in matured oocytes (×15,000).

Table 4.

Number and distribution of mitochondria of in vitro matured buffalo oocytes (in 1 µm2 per oocyte) cultured under low (5%) or high oxygen tensions (20%)

As shown in Fig. 8, the fluorescence intensity of mitochondrial activity was similar in control, BCB+, and BCB− oocytes. In addition, the groups of oocytes cultured under low oxygen levels revealed an insignificant elevation in mitochondrial activity compared with their counterparts cultured under high oxygen levels, except for BCB+ oocytes.

Figure 8.

Fluorescence intensity of intracellular mitochondrial activity in mature buffalo oocytes cultured under low (5%) or high oxygen tensions (20%) during in vitro maturation. Bars with different superscripts (a–c) indicate significant difference at P ≤ 0.05.

Reactive oxygen species levels

The level of ROS was measured to detect the effect of oxygen tension on the microenviroment of oocytes and subsequent effect on cytoplasmic and nuclear maturation of buffalo BCB+ and BCB− COCs. The ROS level in the cytoplasm of BCB− oocytes cultured under 20% oxygen was significantly (P ≤ 0.05) higher than in BCB− group cultured under 5% oxygen (Fig. 9).

Figure 9.

Fluorescence intensity of reactive oxygen species (ROS) of matured buffalo oocytes cultured under low (5%) or high oxygen tensions (20%) during in vitro maturation. Bars with different superscripts (b, c) indicate significant difference at P ≤ 0.05.

Gene expression

Maturation of COCs is dependent on cytoplasmic, nuclear and molecular changes. Therefore, four genes were selected according to their biological roles and their profile link with subcellular, cytoplasmic and nuclear measurements performed during COC maturation. The MAPK14 gene is involved in oocyte maturation (Fan and Sun, Reference Fan and Sun2004; Villa-Diaz and Miyano, Reference Villa-Diaz and Miyano2004), while SOD2 plays a major role in cellular defence mechanisms against oxidative stress (Balasubramanian et al., Reference Balasubramanian, Son, Kumar, Ock, Yoo, Im, Choe and Rho2007; Ma et al., Reference Ma, Chen and Tzeng2017). Additionally, CPT2 encodes a protein that oxidizes long-chain fatty acids (FAs) in mitochondria (Spikings et al., Reference Spikings, Alderson and John2007; Downs et al., Reference Downs, Mosey and Klinger2009) and NFE2L2 induces the expression of a variety of antioxidant response element-dependent genes to regulate physiological and pathophysiological outcomes after exposure to different stressors (Ma et al., Reference Ma, Liang, Sun, Qiu, Lin, Ge, Jueraitetibaike, Xie, Zhou, Huang, Wang and Chen2018; Khadrawy et al., Reference Khadrawy, Gebremedhn, Salilew-Wondim, Taqi, Neuhoff, Tholen, Hoelker, Schellander and Tesfaye2019).

According to the oxygen level, the transcript abundance of MAPK14 was lower in all groups of oocytes cultured under high oxygen tension than those cultured under low oxygen tension. The gene expression profile of MAPK14 was increased (P ≤ 0.05) in control and BCB+ oocytes compared with BCB−. BCB− oocytes recorded the lowest (P ≤ 0.05) expression profile of MAPK14 under high oxygen tension (Fig. 10).

Figure 10.

Gene expression profiles of MAPK14, SOD2, CPT2 and NFE2L2 in matured buffalo oocytes cultured under low (5%) or high oxygen tensions (20%) during in vitro maturation. Bars with different superscripts (a–d) indicate significant difference at P ≤ 0.05.

There were no significant differences in the SOD2 expression profile (Fig. 9) among all experimental groups, although oocytes cultured under low oxygen tension had a higher expression than those cultured under high oxygen tension.

The expression profile of the CPT2 gene (Fig. 9) was increased in all groups of oocytes cultured under low compared with high oxygen tensions. The highest (P ≤ 0.05) expression of CPT2 was recorded in the BCB− group cultured under low oxygen levels. In contrast, BCB− oocytes cultured under high oxygen levels expressed the lowest transcript abundance of CPT2 during in vitro maturation.

The expression profile of NFE2L2 (Fig. 9) was upregulated (P ≤ 0.05) in all groups of oocytes cultured under high oxygen tension compared with their counterparts cultured under low oxygen tension. The BCB+ oocytes cultured under high oxygen tension expressed the highest (P ≤ 0.05) profile of the NFE2L2 gene while BCB− oocytes cultured under low oxygen tension recorded the lowest expression of this gene.

Discussion

Despite the progress in buffalo IVP (Madan et al., Reference Madan, Singla, Chauhan and Manik1994a) and embryo transfer (Madan et al., Reference Madan, Chauhan, Singla and Manik1994b), the efficiency of IVP is still very low, in terms of transferable embryos rate (TE) and development to term (Madan et al., Reference Madan, Das and Palta1996). Seasonal variations in embryo development rate have been linked to alterations in oocyte quality in Italian buffaloes (Di Francesco et al., Reference Di Francesco, Boccia, Campanile, Di Palo, Vecchio, Neglia, Zicarelli and Gasparrini2011). The quality of the oocyte is one of the main factors that affects embryo yield, while culture conditions influence embryo quality (Rizos et al., Reference Rizos, Ward, Duffy, Boland and Lonergan2002). The results of the current study inferred no effect of oxygen tension level (20% vs. 5%) and oocyte quality (BCB+ vs. BCB−) on cumulus expansion rate. However, nuclear maturation rate was increased in all groups of oocytes cultured under low oxygen tension (5%) compared with groups cultured under high oxygen tension (20%). Similarly, Kumar et al. (Reference Kumar, Verma, Kumar, De, Kumar and Datta2015) have reported the enhancement of maturation and blastocyst rates at low oxygen levels (5%). In vitro culture of preimplantation stages at low oxygen tension has improved the rate of nuclear maturation, embryo quality, and development to the blastocyst stage in different species (Farrell and Foote, Reference Farrell and Foote1995; Hashimoto et al., 2000; Van Soom et al., Reference Van Soom, Yuan, Peelman, de Matos, Dewulf, Laevens and de Kruif2002; Ciray et al., Reference Ciray, Aksoy, Yaramanci, Karayaka and Bahceci2009).

In the present study, BCB− oocytes recorded the lowest maturation rate during in vitro maturation under high oxygen tension. It has been established that BCB− oocytes had lower developmental competence than BCB+ oocytes in buffalo (Manjunatha et al., Reference Manjunatha, Gupta, Devaraj, Ravindra and Nandi2007), bovine (Alm et al., Reference Alm, Torner, Loehrke, Viergutz, Ghoneim and Kanitz2005), camel (Fathi et al., Reference Fathi, Ashry, Salama and Badr2017), equine (Mohammadi-Sangcheshmeh et al., Reference Mohammadi-Sangcheshmeh, Held, Ghanem, Rings, Salilew-Wondim, Tesfaye, Sieme, Schellander and Hoelker2011), sheep (Catalá et al., Reference Catalá, Izquierdo, Uzbekova, Morató, Roura, Romaguera, Papillier and Paramio2011), goat (Rodríguez-González et al., Reference Rodríguez-González, López-Bejar, Izquierdo and Paramio2003), and mice (Wu et al., Reference Wu, Liu, Zhou, Lan, Han, Miao and Tan2007). The reduction of nuclear maturation rate of BCB− oocytes under high oxygen tension may be due to chromosomal defects that have occurred under oxidative stress (Tarin et al., Reference Tarin, Vendrell, Ten, Blanes, van Blerkom and Cano1996; Choi et al., Reference Choi, Banerjee, Falcone, Bena, Agarwal and Sharma2007; Tamura et al., Reference Tamura, Takasaki, Miwa, Taniguchi, Maekawa, Asada, Taketani, Matsuoka, Yamagata, Shimamura, Morioka, Ishikawa, Reiter and Sugino2008), but this needs to be confirmed. The higher rates of nuclear maturation and cortical granule migration in BCB+ oocyte support the idea for increased nuclear and cytoplasmic maturity of this group of oocytes. Conversely, delayed migration of cortical granules in BCB− could be due to cytoskeleton disorganization (Silva et al., Reference Silva, Rodriguez, Galuppo, Arruda and Rodrigues2013; Mao et al., Reference Mao, Lou, Lou, Wang and Jin2014) that resulted in unsynchronized events of nuclear, as well as cytoplasmic, maturation in buffalo BCB− oocytes under high oxygen tension. The low maturity of BCB− was also linked to the reduced expression of MAPK14 under high oxygen tension. In support of our results, MAPK activity was reduced in porcine oocytes cultured under thermal stress and this subsequently led to decreased viability (Yen et al., Reference Yen, Tseng, Chuang, Chen and Ju2014). The MAPK family has a crucial role in the meiotic progression of oocytes from different mammalian species (Fan and Sun, Reference Fan and Sun2004; Villa-Diaz and Miyano, Reference Villa-Diaz and Miyano2004; Baatout et al., Reference Baatout, Jaussi, Michaux, Buset, Schoonjans and Jacquet2007; Baluch and Capco, Reference Baluch and Capco2008; Torner et al., Reference Torner, Ghanem, Ambros, Hölker, Tomek, Phatsara, Alm and Sirard2008; Uzbekova et al., Reference Uzbekova, Salhab, Perreau, Mermillod and Dupont2009), as indicated by its involvement in correct chromosome segregation and meiotic spindle stabilization (Baluch and Capco, Reference Baluch and Capco2008).

Surprisingly, downregulation of MAPK14 was coupled with increased ROS levels in BCB− oocytes under high oxygen tension. Interestingly, ROS accumulation could be a key factor that causes low maturation rate in this group of oocytes. This result is supported by early studies that reported an increased incidence of chromosomal errors and disorganization of meiotic spindles in oocytes that had a high level of ROS (Tarin et al., Reference Tarin, Vendrell, Ten, Blanes, van Blerkom and Cano1996; Choi et al., Reference Choi, Banerjee, Falcone, Bena, Agarwal and Sharma2007; Tamura et al., Reference Tamura, Takasaki, Miwa, Taniguchi, Maekawa, Asada, Taketani, Matsuoka, Yamagata, Shimamura, Morioka, Ishikawa, Reiter and Sugino2008). However, studies on MAPK14 have reported its upregulation under increased levels of ROS, which represents an essential trigger for oocyte maturation (Shkolnik et al., Reference Shkolnik, Tadmor, Ben-Dor, Nevo, Galiani and Dekel2011). These results supported our hypothesis that events controlling maturation (cortical granules migration, nuclear maturation, and genes regulating these events) are dysregulated in buffalo BCB− oocytes under high oxygen tension.

Nuclear erythroid 2-related factor 2 (NFE2L2, also know as NRF2) is an important transcriptional factor that regulates antioxidant defence mechanism, while its downregulation decreased mitochondrial activity and finally compromised cellular proliferation (Khadrawy et al., Reference Khadrawy, Gebremedhn, Salilew-Wondim, Taqi, Neuhoff, Tholen, Hoelker, Schellander and Tesfaye2019). Our results indicated the upregulation of NFE2L2 in groups of oocytes cultured under high oxygen tension coupled with reduced mitochondrial activity, which reflected the enhanced molecular response of oocytes to increased oxygen level. The expression of NFE2L2 and level of ROS were elevated in bovine granulosa cells cultured under heat shock (Alemu et al., Reference Alemu, Pandey, Salilew-Wondim, Gebremedhn, Neuhof, Tholen, Holker, Schellander and Tesfaye2018). Increased expression of NFE2L2 could be an attempt by the oocyte to protect itself from any possible deleterious effects of high levels of ROS on meiotic progression. This idea was confirmed in a previous study that demonstrated the reduction of meiotic defects in aged oocytes after overexpression of NFE2L2 (Ma et al., Reference Ma, Liang, Sun, Qiu, Lin, Ge, Jueraitetibaike, Xie, Zhou, Huang, Wang and Chen2018). Interestingly, the expression of NFE2L2 was higher in BCB+ than BCB− oocytes under high oxygen tension. This supported the idea that BCB+ oocytes were more competent than BCB− at the molecular level and that this ensured meiotic progression and subsequent embryonic development (Torner et al., Reference Torner, Ghanem, Ambros, Hölker, Tomek, Phatsara, Alm and Sirard2008). Moreover, this added a new explanation for the increased developmental competence of BCB+ by upregulating antioxidant defence genes in response to any change in their microenvironment.

The redox balance of mammalian cells is regulated by several antioxidant enzymes such as peroxiredoxin (PRDX), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GPx) (Cetica et al., Reference Cetica, Pintos, Dalvit and Beconi2001; Rizzo et al., Reference Rizzo, Roscino, Binetti and Sciorsci2012). Surprisingly, the transcript abundance of SOD2, which is considered to be the downstream gene of NFE2L2, did not show clear changes under high oxygen tension. However, previous studies have reported that embryos developed under low O2 levels had higher expression profiles for MnSOD and PRDX5 compared with their counterparts developed under high O2 levels to neutralize excess ROS generated as a by-product of preimplantation metabolic activity (Balasubramanian et al., Reference Balasubramanian, Son, Kumar, Ock, Yoo, Im, Choe and Rho2007; Ma et al., Reference Ma, Chen and Tzeng2017). One factor behind the observed molecular alteration in buffalo oocytes could be the species effect on gene expression profiles, as indicated in previous studies (Combelles et al., Reference Combelles, Gupta and Agarwal2009; Kandil et al., Reference Kandil, Ghanem, Abdoon, Hölker, Phatsara, Schellander and Tesfaye2010; Ghanem and El-Sayed, Reference Ghanem and El-Sayed2012). For example, the transcripts of the cell cycle namely MPHOSPH9 (Kandil et al., Reference Kandil, Ghanem, Abdoon, Hölker, Phatsara, Schellander and Tesfaye2010) and calcium ion binding such as ANXA2 (Ghanem and El-Sayed, Reference Ghanem and El-Sayed2012) were expressed in buffalo oocytes on an opposite pattern compared with that of their bovine counterparts during in vitro maturation.

Enhanced nuclear maturation of buffalo BCB+ was coupled with an increased rate of centrally located mitochondria and this was in accordance with data reported in bovine (Silva et al., Reference Silva, Rodriguez, Galuppo, Arruda and Rodrigues2013). Recently, porcine BCB+ oocytes recorded a higher rate of diffuse distribution of mitochondria compared with BCB− oocytes after IVM as an indicator of enhanced cytoplasmic maturation (Fu et al., Reference Fu, Ren, Liu, Ma, An, Yang, Ma, Zhang, Guo, Guo, Zhu and Bai2015). The change in mitochondrial distribution linked with enhanced nuclear maturation of BCB+ oocytes could either reflect normal cytoplasmic changes that orchestrate progression to maturation or the enhanced metabolic activity required for the maturation process. However, additional investigations are needed to confirm this idea. Our results indicated the enhancement of mitochondrial fluorescence intensity in groups of oocytes cultured under low oxygen tension compared with their counterparts that were cultured under high oxygen tension, however there was no difference between BCB+ and BCB− oocytes. Mitochondria are maternally inherited and represent the primary source of ATP in the oocyte (Van Blerkom, Reference Van Blerkom2011). Moreover, mitochondrial activity reflects the process of energy production during preimplantation and is relevant to IVP success (Krisher et al., Reference Krisher, Brad, Herrick, Sparman and Swain2007; Van Blerkom, Reference Van Blerkom2011; Ma et al., Reference Ma, Chen and Tzeng2017; García-Martínez et al., Reference García-Martínez, Sánchez Hurtado, Gutiérrez, Sánchez Margallo, Romar, Latorre, Coy and López Albors2018; Belli et al., Reference Belli, Zhang, Liu, Donjacour, Ruggeri, Palmerini, Nottola, Macchiarelli and Rinaudo2019).

In parallel, the number of mitochondria and lipid levels tend to increase in oocytes cultured under low, compared with high, oxygen tension, which indicated the ability of these oocytes to maintain the sufficient energy production required for nuclear progression (Van Blerkom, Reference Van Blerkom2011; Ma et al., Reference Ma, Chen and Tzeng2017; Belli et al., Reference Belli, Zhang, Liu, Donjacour, Ruggeri, Palmerini, Nottola, Macchiarelli and Rinaudo2019). Recently, it has been demonstrated that mice embryos when developed under 20% O2 had fewer mitochondria and abnormal mitochondria shapes compared with those under 5% O2 (Ma et al., Reference Ma, Chen and Tzeng2017; Belli et al., Reference Belli, Zhang, Liu, Donjacour, Ruggeri, Palmerini, Nottola, Macchiarelli and Rinaudo2019). However, the central distribution and fluorescence intensity of oocyte lipid droplets were, to some extent, higher in BCB− oocytes under low or high oxygen tension compared with the other experimental groups. In another study conducted in bovine, lipid droplets were more localized in the inner cytoplasm than the periphery of the oocytes without changing lipid droplet density after treatment with l-carnitine (Chankitisakul et al., Reference Chankitisakul, Somfai, Inaba, Techakumphu and Nagai2013). The ability of BCB− oocytes to modulate intracellular localization and activity of mitochondria, as well as lipid, could be associated with their low developmental competence. In support of this idea, our data demonstrated increased transcript abundance of the CPT2 gene in all groups of oocytes cultured under low oxygen tension, and the highest profile was observed in the BCB− group linked with increased mitochondrial activity. The increased activity of mitochondrial and expression of mitochondrial replication factor during IVM of BCB− oocytes were reported by Torner et al., (Reference Torner, Ghanem, Ambros, Hölker, Tomek, Phatsara, Alm and Sirard2008). While, Spikings et al. (Reference Spikings, Alderson and John2007) have demonstrated lower copies of mtDNA and a delay in its replication in BCB− because of delayed onset of expression of their nuclear-encoded replication factors during IVM. The induction of meiotic progression was enhanced with the increased mitochondrial metabolic activity of FAs through β-oxidation (Downs et al., Reference Downs, Mosey and Klinger2009). Therefore, it seems that low oxygen tension provides a favourable condition for oocytes to increase β-oxidation and subsequently increased mitochondrial activity by upregulation of CPT2. However, high oxygen tension increased the level of ROS that downregulated genes orchestrating mitochondrial activity and finally compromised nuclear maturation in BCB− oocytes.

In conclusion, the results of the current investigation have demonstrated an increase in the maturation rate of buffalo oocytes cultured under low oxygen tension. Conversely, nuclear maturation (progression to meiotic stages) of buffalo BCB− oocytes was reduced and this was coupled with cytoplasmic (cortical granules migration, distribution of mitochondria and lipid) and molecular alterations (reduced expression of MAPK14 and CPT2 genes) under high oxygen tension.

Financial support

This work was partially funded by a research grant financed by Cairo University project (no. 57/16).

Conflict of interest

All authors of this study declared no conflict of interest.

Ethical standards

This study did not require any ethical approval.

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Figure 0

Table 1. List of primers used for quantitative real-time PCR analysis

Figure 1

Table 2. Cumulus expansion and nuclear maturation rate of buffalo oocytes cultured under 5% and 20% oxygen tensions during in vitro maturation

Figure 2

Figure 1. Cumulus expansion and nuclear maturation rate of buffalo oocytes cultured under 5% and 20% oxygen tension during in vitro maturation. Bars with different superscripts (a–c) indicate significant difference at P ≤ 0.05. Cumulus expansion rate: numbers of oocyte that have completely or partially expanded in cumulus layers/total numbers of cultured oocytes. Nuclear maturation rate (%): oocytes that have reached telophase and MII stages.

Figure 3

Table 3. Cortical granules distribution (migrated and non-migrated) and numbers in vitro matured buffalo oocytes cultured under low (5%) or high oxygen tensions (20%)

Figure 4

Figure 2. Percentage of cortical granules distribution (migrated and non-migrated) in buffalo matured oocytes cultured under low (5%) or high oxygen tension (20%) during in vitro maturation. Bars with different superscripts (a–c) indicate significant difference at P ≤ 0.05. Migrated: moved peripherally beneath the plasma membrane. Non-migrated: diffused in the cytoplasm.

Figure 5

Figure 3. Representative photographs of the cortical granules of in vitro matured Egyptian buffalo oocytes taken with a transmission electron microscope (×6000).

Figure 6

Figure 4. Percentage of lipid droplet distribution (peripheral and central) of buffalo mature oocyte cultured under low (5%) or high oxygen tensions (20%) during in vitro maturation. Bars with different superscripts (a–c) indicate significant difference at P ≤ 0.05. Central: located in the centre of the cytoplasm. Peripheral: moved peripherally beneath the plasma membrane.

Figure 7

Figure 5. Fluorescence intensity of intracellular lipid content per buffalo mature oocytes cultured under low (5%) or high oxygen tensions (20%) during in vitro maturation. Bars with different superscripts (a–c) indicate significant difference at P ≤ 0.05.

Figure 8

Figure 6. Percentage of mitochondria distribution (peripheral and central) of mature buffalo oocytes cultured under low (5%) or high oxygen tensions (20%) during in vitro maturation. Central: diffused in cytoplasm. Peripheral: moved peripherally beneath the plasma membrane.

Figure 9

Figure 7. Electron micrograph of Egyptian buffalo oocytes showing oval (OM), round (RM), vacuolated (VM), distorted mitochondrial membrane (DMM) and deformed (DM) forms in matured oocytes (×15,000).

Figure 10

Table 4. Number and distribution of mitochondria of in vitro matured buffalo oocytes (in 1 µm2 per oocyte) cultured under low (5%) or high oxygen tensions (20%)

Figure 11

Figure 8. Fluorescence intensity of intracellular mitochondrial activity in mature buffalo oocytes cultured under low (5%) or high oxygen tensions (20%) during in vitro maturation. Bars with different superscripts (a–c) indicate significant difference at P ≤ 0.05.

Figure 12

Figure 9. Fluorescence intensity of reactive oxygen species (ROS) of matured buffalo oocytes cultured under low (5%) or high oxygen tensions (20%) during in vitro maturation. Bars with different superscripts (b, c) indicate significant difference at P ≤ 0.05.

Figure 13

Figure 10. Gene expression profiles of MAPK14, SOD2, CPT2 and NFE2L2 in matured buffalo oocytes cultured under low (5%) or high oxygen tensions (20%) during in vitro maturation. Bars with different superscripts (a–d) indicate significant difference at P ≤ 0.05.