Hydrogen peroxide concentration and DNA fragmentation of buffalo oocytes matured in sericin-supplemented maturation medium

S. Gustina, N.W.K. Karja, H. Hasbi, M.A. Setiadi & I. Supriatna 1 Division of Animal Science, Faculty of Animal Science and Fisheries, Sulawesi Barat University, Majene 91412, Sulawesi Barat, Indonesia 2 Department of Animal Production, Faculty of Animal Science, Hasanuddin University, Makassar 90245, Sulawesi Selatan, Indonesia 3 Division of Reproduction and Obstetrics, Department of Veterinary Clinic, Reproduction, and Pathology, Bogor Agricultural University, Bogor 16680, Jawa Barat, Indonesia


Introduction
In buffalo, the efficiency of in vitro embryo production (IVEP) is still low (Neglia et al., 2003;Drost, 2007).Approximately only 20% of buffalo oocytes that are fertilized in vitro form blastocysts (Gasparrini, 2002;Nandi et al., 2002;Manjunatha et al., 2008).This inefficiency in buffalo IVEP could be associated with the quality of the oocytes at the beginning of the maturation process, although oocytes were selected based on the compaction of cumulus cells and the homogeneity of ooplasm (Chauhan et al., 1998).
One of the critical factors that could affect the quality of oocytes matured in vitro is the oxygen tension under which oocytes are cultured.In vitro culture environments differ from in vivo conditions.The oxygen tension in in vitro culture systems (≈ 20%) is often higher than in the female reproductive tract (3% to 9%) (Rocha-Frigoni et al., 2016).This condition is believed to possibly induce the generation of reactive oxygen species (ROS), such as superoxide anions, hydroxyl radicals and hydrogen peroxide (H 2 O 2 ) (Morado et al., 2009).Reactive oxygen species can modify cell function and endanger the survival of cells that cause fragmentation of DNA, protein oxidation and lipid peroxidation (Yang et al., 1998).High oxygen tension during in vitro maturation and culture might contribute to decreased oocyte quality (Hashimoto et al., 2000) and alter the development of mammalian embryos (Karja et al., 2004).Several enzymatic and non-enzymatic antioxidant mechanisms have been developed that protect oocytes and embryos against the harmful effects of ROS (Johnson & Nasr-Esfahani, 1994).Antioxidants have the roles that they can i) prevent the formation of ROS, ii) inactivate oxidants and iii) possibly limit the deleterious effects of oxidants or repair oxidative damage (Agarwal et al., 2004).
Sericin is a family of proteins that covers fibroin, and as the glue in silkworm cocoons (Takahashi et al., 2003).Sericin is known to have a skin moisturizing and antiwrinkle action owing to its high serine content (Engel et al., 1987).Currently, sericin is used for various purposes, including functional biomaterials (Altman et al., 2003;Cao & Zhang, 2016), pharmacological purposes (Prommuak et al., 2008), medical uses (Prasong, 2011;Kunz et al., 2016), and food supplements (Zhang, 2002).Sericin is a high molecular weight protein with adhesive properties (Aramwit et al., 2010) and is derived from silkworm cocoons (Dash et al., 2008).Sericin has ROS-scavenging, anti-tyrosinase, anti-elastase (Chlapanidas et al., 2013) and antibacterial properties (Sarovart et al., 2003).It can prevent cell death and supports the growth of cells (Agarwal et al., 2004), increasing the growth of fibroblast cells (Aramwit et al., 2009), and stimulates mammalian cell proliferation (Terada et al., 2002).Sato et al. (2011) explained that sericin promotes growth in a variety of cells and affects mitosis in mammalian cells through various signalling pathways.Furthermore, Aramwit et al. (2009) reported that with its high methionine content, sericin can promote the fastest growth rate of fibroblast cells.Methionine increases the accumulation of extracellular matrix components such as collagen type I and activates matrix metalloproteinases-9 as an active part in extracellular matrix modelling.Kato et al. (1998) demonstrated the antioxidant action of sericin by showing it suppresses in vitro lipid peroxidation and inhibits tyrosinase activity in rat brains.Previous studies by Chlapanidas et al. (2013) on in vitro stimulated peripheral blood mononuclear cells showed that sericin can serve as an ROS-scavenger.Following its addition to the culture medium for embryo production in vitro, sericin increased the percentage of maturation and in vitro fertilization of sheep oocytes in a medium without BSA (Yasmin et al., 2015), and increased the quality and pre-implantation development of bovine embryos that were cultured individually (Isobe et al., 2012).However, the role of sericin in DNA fragmentation or the concentration of H 2 O 2 in oocytes matured in vitro has not been studied.Therefore, the objective of this study was to evaluate DNA fragmentation and the concentration of H 2 O 2 of buffalo oocytes matured in maturation medium supplemented with sericin.
The concentrations of H 2 O 2 of matured oocytes were measured using DCHFDA (Sigma) as described by Karja et al. (2006) with minor modifications.Oocytes in each group were incubated for 15 min in maturation medium containing 10 μM DCHFDA, and then washed in fresh medium before being placed on glass slides and covered with cover slips.Fluorescence emissions were recorded with a digital camera (Zeiss AxioCam HRc, Germany) attached to a fluorescence microscope (Zeiss Axio Imager A2) after excitation at 480 nm and 510 nm emission.The fluorescent image was converted to TIFF files using Adobe Photoshop CS3 (Adobe Systems Inc., San Jose, CA), then analysed with ImageJ software 1.47 (Sun Microsystems, Inc., California, USA).The fluorescent image was measured by counting the number of pixels after colour inversion.The fluorescence intensity represents the intracellular H 2 O 2 concentration.
Fragmentation of DNA and nuclear status of matured oocytes in each group were analysed using the combined technique for nuclear staining and TUNEL (in situ cell death detection system) (Roche Diagnostics Corporation, Indianapolis, IN, USA) (procedure modified after those described by Wongsrikeao et al. (2004)).Oocytes were fixed overnight at 4 °C in 3.7% (wt/vol) paraformaldehyde diluted in PBS.After overnight fixation, the oocytes were washed four times in PBS, which contained 3% (w/v) polyvinyl alcohol (PVA) and permeabilized in 0.5% (v/v) Triton-X100 for 1 hour, and then incubated in a blocking solution (PBS + 10 mg/ml BSA) overnight at 4 °C.After being washed in PBS-PVA, positive controls and all treated oocytes were incubated in fluorescein conjugated dUTP and TdT (TUNEL reagent) at 38.5 °C for 1 hour in the dark.
As positive controls, one to two oocytes per TUNEL analysis were incubated in 1000 IU/mL deoxyribonuclease I (DNase I) (Sigma) for 20 min.Negative controls were incubated in fluorescein dUTP without TdT.After TUNEL, oocytes were washed three times in PBS-PVA, and later stained with 50 ug/mL propidium iodide (PI) for 20 min to label all nuclei.Oocytes were washed extensively in blocking solution and placed on a glass slide and covered with a glass cover.The oocytes were examined under a fluorescence microscope (Zeiss Axio Imager A2) using excitation at a wavelength of 488 nm and 568 nm to detect the reaction of TUNEL and PI.The images were captured with a digital camera (Zeiss AxioCam HRc, Germany) (Figure 1).To assess the relationship between nuclear status and DNA fragmentation, chromatin staining of oocytes was used, classify them based on configuration from GV to MII.Those oocytes with diffusely stained cytoplasm characteristics of non-viable cells and those in which chromatin was not visible were excluded from DNA fragmentation analyses.

Discussion
During maturation and culturing, various factors can affect the quality and development of oocytes and embryos, especially environmental conditions.Oxygen tension during the culture process is influential on embryo development.Culture conditions under high oxygen tension have a negative effect that can induce the generation of ROS (Rocha-Frigoni et al., 2016).ROS can alter most types of cellular molecules, and block the development of pre-implantation embryos (Guerin et al., 2001).
In this study, the addition of 0.05% sericin significantly increased the percentage of meiotic maturation of buffalo oocytes compared with the (-BSA) and (+BSA) groups.These data are consistent with the findings of Isobe et al. (2012) that the addition of 0.5% sericin can improve the quality and preimplantation development of bovine embryos.In sheep, maturation and fertilization rates can be improved with the addition of 0.1% sericin (Yasmin et al., 2015).On the other hand, the authors found that the level of nuclear maturation in the high concentration sericin group (0.25%) showed a significant decrease, possibly owing to a toxic effect of sericin.According to Aramwit et al. (2010), sericin can increase collagen production, depending on the concentration, but high concentrations can cause cellular toxicity.
Furthermore, in this study, 0.05% sericin supplementation reduced the concentrations of intracellular H 2 O 2 compared with the (-BSA) group.Dash et al. (2008) reported that sericin reduced levels of catalase, lactate dehydrogenase and the activity of malondialdehyde (markers of oxidative stress) in fibroblast cells exposed H 2 O 2 .Isobe et al. (2012) reported that sericin may have a unique antioxidant potential.The mechanism of the antioxidant effect of sericin is chelation of trace elements, because of its high content of hydroxyl amino acids (serine and threonine) (Kato et al., 1998).
Hydrogen peroxide penetrates biological membranes (Silva & Coutinho, 2010).The mechanism of damage by H 2 O 2 in cultured oocytes involves ROS derivatization.Reactive oxygen species can spread and pass through cell membranes and alter cellular molecules, such as proteins, lipids and nucleic acids, resulting in alterations in the mitochondria, embryo cell blockage, depletion of adenosine triphosphate (ATP) and DNA fragmentation (Guerin et al., 2001).DNA fragmentation indicates apoptosis, in which the generation of ROS can result in DNA strand breaks that arrest the cell cycle or even cause cell death (Fahrudin et al., 2002).Therefore, the authors also observed the index of DNA fragmentation in oocyte nuclei matured with sericin.Apoptosis is a normal process during preimplantation embryo development, which may be due to environmental stress and chromosome abnormalities (Matwee et al., 2000).Apoptosis is characterized by loss of phospholipid balance in the plasma membrane, chromatin condensation, DNA fragmentation and blebbing of the plasma membrane.In fragmentation, the cell becomes condensed and divides into several fragments, causing cytoplasmic condensation, condensed nuclei (Wyllie et al., 1980), and DNA breakdown, resulting in DNA fragments of 180 -200 base pairs (Elmore, 2007).
This study demonstrated that although sericin can enhance the number of oocytes that reach MII, there were no significant differences in H 2 O 2 concentration or the total of oocytes undergoing DNA fragmentation in all treatment groups.Although the reasons for this finding are currently unclear, the authors presume that it may be due to the effect of glutathione (GSH), which is a natural intracellular antioxidant.Levels of intracellular GSH increase during the oocyte maturation process in the ovary, and peak when oocytes reach MII (Deleuze & Goudet, 2010).Similar to other species, buffalo oocytes can synthesize GSH de novo during IVM (Gasparrini et al., 2003).Nevertheless, the results of this study show that the addition of sericin to maturation media may serve as a replacement for BSA to increase the maturation rate, and reduce the concentration of H 2 O 2 and the proportion of DNA fragmentation.Bovine serum albumin is the main soluble protein in the circulatory system and has many physiological functions.BSA serves as a depot protein and as a transport protein for a variety of compounds (Papadopoulou et al., 2005).It is generally used as a supplement in culture medium.Although BSA can protect cells and has many advantages, there are sanitary risks such as disease transmission, and it may serve as a contamination vector (Thibier, 2006).BSA is not only expensive in the long term, but it leads to enhanced risk of many zoonotic diseases (Cao & Zhang, 2016).This finding is in agreement with other studies, which were reported by Yasmin et al. (2015) in sheep oocytes, and Terada et al. (2002) in several mammalian cell lines in which sericin was used to accelerate cell proliferation.Sericin may serve as an alternative supplement in serum-free media for cell culture (Tunma et al., 2013).Yanagihara et al. (2006) reported that sericin is a powerful alternative to the production of the adenoviral vector in human cells.

Conclusion
In conclusion, the current research findings suggest that supplementation of 0.05% sericin in the maturation medium enhances meiotic maturation of buffalo oocytes.Additionally, sericin can replace BSA as a supplement in serum-free medium, which at 0.05% may decrease the concentration of intracellular H 2 O 2 in buffalo oocytes.Further research is warranted to evaluate the effects of sericin supplementation in the quality and development of buffalo embryos.

Table 1
Meiotic maturation of buffalo oocytes with supplementation of sericin in maturation medium a, b, c ) in the same column differ significantly (P ≤0.05) Ser: sericin; BSA: bovine serum albumin; AI/TI: anaphase I and telophase I; GV: germinal vesicle; GVBD: germinal vesicle breakdown; MI: metaphase I; MII: metaphase II; No: number of oocytes

Table 2
Nuclear DNA fragmentation of buffalo oocytes matured with sericin