Effect of Energy Substrate Dynamics on Mitochondrial Activity and Oxidative Stress Levels of in vitro Maturing Bovine Oocytes

This study was conducted to explore the effect of energy substrates in the culture medium during in vitro maturation of bovine oocytes. A modified TCM199 medium (M-7528) was used to mature bovine oocytes in vitro. Oocytes were supplemented with different pyruvate (0.1, 0.2, 0.4 mM) and glucose (1.5, 5.6, 20.0 mM) concentrations for 48 hours at 38.5 °C under 5% CO2 atmosphere with 95% humidity. Their maturity was checked at 24 and 48 hours. After 48 hours, the denuded oocytes were stained with fluorescent dye JC-1 and avidin-FITC. Fluorescent dye JC-1 is a membrane permeable to the cell and would indicates membrane activity or its organization. Fluorescence intensity of avidin-FITC determination using corrected total cell fluorescence (CTCF) expressed oxidative stress level. There is a significant contribution of energy substrates towards oocyte maturation. Pyruvate at 0.2 mM produced mature oocytes with a diameter of ≥ 120 μm, promoted oocytes maturation to metaphase II (MII) stage faster and reduced cell’s oxidative stress levels. In comparison, 5.6 mM glucose is the optimum concentration for glucose to reduce cell stress level. Unfortunately, this concentration only produced mature oocytes with a small diameter of up to 116 μm. All changes were significant at the level of p < 0.05. As a conclusion, pyruvate at 0.2 mM is the optimum concentration for in vitro maturation after taking cell’s stress level into consideration.

pyruvate (Nandi, Kumar, Manjunatha, Ramesh, & Gupta, 2008). Glucose helps cumulus-oocyte-complex (COCs) to mature (Pries & Gardner, 2005), and is essential for cellular functions during oocyte maturation (Sutton-McDowall, Gilchrist, & Thompson, 2010). Pyruvate is a main energy substrate used directly for oocyte maturation and resumption of meiosis (Biggers, Whittingham, & Donahue, 1967;Roberts et al., 2004;Pries, Seidel, & Gardner, 2005;Sutton-McDowall, Gilchrist, & Thompson, 2010). This metabolic activity plays a crucial role in oocyte quality because glycolytic activity in mature oocytes is correlated with increased embryo development (Krisher, 2004). Thus, level of both substrates can have a profound effect on oocyte maturation and adequate levels of glucose and pyruvate are important for progression of meiosis to metaphase II. Hence, understanding energy substrate metabolism of the oocyte throughout IVM may aid in optimizing maturation condition.
Mitochondria are double membrane organelles that play a fundamental role in the cell and mitochondrial dysfunction. It has been linked with several pathologies, including infertility (Wallace, 1999;Ramalho-Santos et al., 2009). Mitochondria's vital role in the oocytes metabolism is to provide ATP for fertilization and preimplantation embryo development. The rate of oocyte respiration depends on two major factors: (i) the efficiency of conversion in the oocyte cytoplasm of metabolic precursors such as glucose to pyruvate, and (ii) the efficiency of the mitochondria matrix in the conversion of pyruvate to ATP (Wilding et al., 2001;Vellila, Gonzalez, Vidal, Izquierdo, & Panamio, 2006;Ramalho-Santos et al., 2009). JC-1 (5,5'6,6'-tetrachloro-1,1'3,3'tetraethylbenzimidazolycarbo cyanine iodide) is a fluorescent dye which accumulates in mitochondria and one of the most specific stains currently used to detect mitochondrial membrane potential for oocytes (Smiley et al., 1991;Wilding et al., 2001;Blerkom & Davis, 2007;Thompson, Lane, & Gilchrist, 2007). Due to this, JC-1 can be used as an indicator of mitochondrial activity.
It is estimated that 85-90% of a cell's oxygen is consumed by mitochondria in oxidative phosphorylation. Due to high usage of oxygen, potentially harmful reactive oxygen species (ROS) such as superoxide radicals, hydroxyl radicals and hydrogen peroxide are generated in abundance (Sugino, 2005(Sugino, , 2006Ramalho-Santos et al., 2009). Past studies have indicated that mitochondria are the major ROS generator, with 0.2-2% of the oxygen taken up by the cell is converted to ROS (Ramalho-Santos et al., 2009).
Oxidative stress occurs as a consequence of the excessive production of reactive oxygen species and impaired of antioxidant defence mechanism. According to Sugino (2005), ROS in oocytes are produced within the follicle, especially during the ovulatory process. Studies have indicated that oxidative stimulation plays an important physiological role in maturation in which it promotes oocyte maturation and follicular wall rupture within the follicle. However, when unregulated the excessive production of ROS may lead to an increased risk of poor oocyte quality (Tamura et al., 2007;Ramalho-Santos et al., 2009). Hence, the present study would like to determine the relationship between oxidative stress level and mitochondrial activity in matured oocytes. Intrafollicular concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG) were used as a sensitive indicator of DNA damage. As a biomarker of oxidative stress, 8-OHdG was measured through the use of avidin conjugated-fluorescein isothiocyanate (FITC) (Struthers, Patel, Clark, & Thomas, 1998).
In summary this study was designed to: (1) determine if COCs diameter is a good reflection of oocyte maturation; (2) determine the optimum glucose and pyruvate concentration for mitochondrial activity in matured oocytes; and (3) determine the optimum glucose and pyruvate concentration for oxidative stress levels in matured oocytes.

Oocyte Collection
Bovine ovaries were obtained at a local abattoir and transported to the laboratory in sterile 0.01 M Phosphate-Buffered Saline (PBS), pH 7.4 at 37 °C. Transportation was done within 3 hours after sacrifice. After the ovaries were washed twice in sterilized PBS solution, cumulus-oocyte complexes (COCs) were obtained from the ovaries by a slicing method. COCs having four to five layers of intact cumulus cells and homogeneous cytoplasm were then rinsed thrice in PBS medium containing 5% (v/v) calf serum, 10,000 IU Penicillin and 10 mg of Streptomycin. They were then used for later experiment.

In vitro Maturation
The IVM medium used was a tissue culture medium 199 (with 25 mM HEPES, Earl's salts, L-glutamine and 2 mg mL -1 sodium bicarbonate; Sigma) modified by the addition of 4 mg mL -1 bovine serum albumin and gentamicin 50 µg mL -1 . Each 10 to 20 oocytes were then cultured in100µL of maturation solution under mineral oil. Ten oocytes per mineral oil drop was incubated for 48 hours at 38.5 °C under 5% CO 2 atmosphere with 95% humidity. Oocytes were checked for maturity at 24 and 48 hours. After 48 hours, oocytes were denuded mechanically from cumulus cells in modified phosphate-buffered saline supplemented with 1 mg/ml hyaluronidase.

JC-1 Staining
The fluorescent dye JC-1 (5,5'6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazolcarbocyanine iodide, Molecular Probes, USA) was used to measure the activity of mitochondria. A stock solution of JC-1 was made at a concentration of 1 mg/ml in dimethyl sulfoxide. Fresh staining solution (10 µg/ml) was prepared by diluting the stock solution in warm (37 °C) culture medium supplemented with 10% calf serum. For each 10 oocytes, 50 µl of fresh JC-1 solution was immediately applied to a slide. They were then placed in an incubator for 30 min. A Partec CyScope fluorescence microscope was used for all experiments. An Argon laser was used to produce the excitation laser line at 472 nm, and emission wavelengths were separated by a 500 nm dichroic mirror followed by analysis in a photomultiplier after further filtering through a 536 nm band pass filter (green emission). Images were processed by using Videology and Image-J software.

Avidin-FITC Staining
Fluorescent assessment of avidin-FITC was used for detection of 8-OHdG (Struthers, Patel, Clark, & Thomas, 1998). The denuded oocytes were washed in PBS after fixing with 2% (w/v) paraformaldehyde (in PBS at pH 7.4) for 15 min. Then, oocytes were washed in warm PBS twice. They were later fixed and permeabilized with ice-cold methanol for 15 min. They were washed twice with PBS and then the oocytes were chemically blocked for 10 min at room temperature in 300 µl PBS containing 10% (w/v) NGS (normal goat serum). The blocking solution was washed off with PBS containing 0.2% (w/v) NGS. For each 10 oocytes, 50 µl of avidin-conjugated FITC solution (suspended 1:200 in PBS) was immediately applied to a slide, which was then incubated for an hour at room temperature. A Partec CyScope fluorescence microscope was used for all experiments. An Argon laser was used to produce the excitation laser line at 470 nm, and emission wavelengths were separated by a 500 nm dichroic mirror followed by analysis in a photomultiplier after further filtering through a 550 nm band pass filter. Images were processed by the Videology and fluorescence intensities were analysed by the Image-J software (Burgess et al., 2010;Gavet & Pines, 2010;Potapova, Sivakumar, Flynn, Li, & Gorbsky, 2011).

Experimental Design
Experiment 1: COCs and oocyte size measurement.
COCs and oocyte diameter was measured using Image-J software after the images were processed by Videology for CyScope microscope connected to an USB CCD camera. A mean of two measurements for each COCs and oocyte diameter was made perpendicular to each other.
Experiment 2: Determination of optimum glucose and pyruvate concentration for mitochondrial organization in matured oocytes.
A minimum of 60 oocytes were matured in each group of IVM media with four different glucose concentrations 0, 1.5, 5.6 and 20.0 mM, respectively (modification of Hashimoto, Minami, Yamada, & Imai, 2000) and four different sodium pyruvate concentrations 0, 0.1, 0.2 and 0.4 mM. Each group had undergone an incubation period and stained with JC-1 fluorescent probes. Mitochondrial organization pattern and activity in matured oocytes were factored-in during statistical analysis.
Experiment 3: Determination of optimum glucose and pyruvate concentration for oxidative stress level in matured oocytes.
A minimum of 60 oocytes were matured in each group of IVM media with four different glucose concentrations 0, 1.5, 5.6 and 20.0 mM, (modification of Hashimoto, Minami, Yamada, & Imai, 2000) and four different sodium pyruvate concentrations 0, 0.1, 0.2 and 0.4 mM. Each group had undergone an incubation period and stained with avidin-FITC fluorescent probes. Corrected total cell fluorescence (CTCF) was used to determine the level of fluorescence in a given region (oocyte) and this fluorescence intensity expressed the oxidative stress level. The method of calculation is, CTCF = Integrated density -(Area of selected cell × Mean fluorescence of background readings) (Burgess et al., 2010;Gavet & Pines 2010;(Burgess et al., 2010;Gavet & Pines, 2010;Potapova, Sivakumar, Flynn, Li, & Gorbsky, 2011).

Statistical Analysis
All oocytes were randomly distributed within each experimental group and each experiment was repeated at least three times. All percentage data were subjected to arc-sine transformation before statistical analysis. Statistical analyses were carried out using Pearson Correlation test and two-way analysis of variance (ANOVA). For the jas.ccsenet. statistical a of P < 0.05

Results
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[Oocyte di used to det played a ro diameter i contribute    Deldar et al., 2011). In this present study, a similar trend of development of glucose was observed. By culturing bovine oocyte (COCs) in medium containing lower concentration (1.5 mM) or higher concentration (20 mM) of glucose, the resumption of meiosis and completion of maturation to metaphase-II stage was vastly reduced. Similar pattern was also observed by Chang, Dale, and Moley (2005) which had reported that maternal diabetes and hyperglycaemia have adversely affected oocyte maturation (Chang et al., 2005). This had led to poor developmental competence.
The present study demonstrated that supplementation of pyruvate 0.1 mM and 0.2 mM in maturation medium significantly reduced the oxidative cell's stress level in oocytes. Addition of 0.4 mM pyruvate in maturation medium had not significantly reduced the oxidative cell's stress level in oocytes compared to control. This results supported recent observation that pyruvate had a bi-functional role both as an energy substrate and as an antioxidant (Nandi, Kumar, Manjunatha, Ramesh, & Gupta, 2008). Hashimoto, Minami, Yamada, and Imai (2000) reported that glucose during oocyte maturation had increased the contents of reactive oxygen species in oocytes, decreased glutathione levels and impaired the developmental competence of oocytes. Furthermore, previous studies have suggested that the toxic effect of ROS generated had caused a negative feedback of cell respiration and disruption of mitochondrial organization. All this had resulted in precocious resumption of nuclear maturation (Barnett, Clayton, Kimura, & Bavister, 1997;Sutton-McDowall, Gilchrist, & Thompson, 2010). Conversely, present study found that addition of glucose in maturation medium had significantly reduced the oxidative cell's stress level in oocytes compared to control maturation medium.
Taken together, these data suggest that supplementation of 0.2 mM pyruvate in maturation medium of bovine oocytes will produce matured oocytes with a diameter ≥ 120 μm. Moreover, the oocyte will not shrink as observed with the addition of glucose. Resumption of meiosis will also occur thus allowing the oocytes to mature to metaphase II (MII) stage much faster. Oocytes supplemented with pyruvate recorded lower oxidative stress levels.
On the other hand, the addition of 5.6 mM glucose in the maturation media would only enhance the oocyte maturation rate but not deliver other additional benefits as done by pyruvate. Further understanding on glucose metabolism during oocyte maturation may lead to improved IVM culture conditions, as well as an involvement in planning of treatment for diabetic or obese women with low fertility (Pasquali et al., 2007).
In conclusion, results obtained in the present study demonstrated that 0.2 mM pyruvate is the optimum concentration for mitochondrial organisation while taking into consideration oxidative stress levels of matured bovine oocyte.