M. Nursadida, W. Widjiati*, I. Mustofa, E. Safitri, S. Susilowati and T. Wahyu Suprayogi
The success of in vitro fertilization and embryo culture depends on the success of in vitro maturation. However, standard culture conditions usually increase reactive oxygen species (ROS), which have been implicated as a major cause for reduced embryonic development. It is well-known that higher than physiological levels of ROS trigger granulosa cell apoptosis and thereby reduce the transfer of nutrients and survival factors to oocytes, leading to apoptosis. This study aimed to determine the optimal timing of oocyte maturation and its relationship to DNA fragmentation. Ovaries were collected from a slaughterhouse and the follicles aspirated. The cumulus oocyte complexes were divided into groups and transferred to a maturation medium, where they were maintained for 18 hours (P1), 22 hours (P2) and 24 hours (P3) to evaluated maturation rate. Matured oocytes were characterized as oocytes that reached the MII stage. Matured oocytes were counterstained with terminal deoxynucleotidyl transferase nick-end labelling (TUNEL). The results showed that the maturity rate of Kacang goat oocytes reached 46% after 18 hours, 77% after 22 hours, and 63% after 24 hours. However, the results showed that the expression of DNA fragmentation in P2 (2.4 ± 0.89) were significantly different from P1 (5.4 ± 2.61) and P3 (9.0 ± 2.12). In conclusion, the optimal timing of in vitro maturation of Kacang goat oocytes is 22 hours.
Key words: IVM; DNA fragmentation; optimal timing; Kacang goat; food production
Biotechnology methods have been developed and used to improve the efficiency of livestock reproduction (Susilowati et al., 2018). In vitro production of embryos refers to the use of laboratory techniques to produce embryos (Asad et al., 2018). The in vitro embryo production (IVEP) method involves three basic steps: in vitro maturation (IVM) of primary oocytes collected from follicles, fertilization of matured secondary oocytes, and culture of potential probable embryos to the blastocyst stage (Avelar et al., 2012). The efficiency of IVEP is limited as it can generate about 30–50% of viable blastocysts (Fernandes-Franca et al., 2020). In vitro maturation (IVM) is the initial step of the IVEP method (Maskura et al., 2020). In the in vitro maturation process, a stable culture system and quality of oocytes are needed as a good source of oocytes.
There are several things that can increase the success of oocyte maturation, depending on the culture condition used, such as hormonal stimulation in the medium, medium supplementation, species characteristics, and time of exposition of maturation (Crocomo et al., 2020; Widjiati et al., 2020). Optimal culture timing is helpful for developmental competence of IVM oocytes (Yu et al., 2016). When culture time is too short, the maturation of the cytoplasm and the nucleus is not synchronized, which affects the subsequent development potential of the embryo (La et al., 2019). A long storage time can reduce the quality of oocytes (Widjiati et al., 2020). After 24 hours of IVM, ovine oocytes showed signs of degeneration, such as ooplasm shrinkage and granulation, and a disorganization in mitochondrial distribution (Maximo et al., 2012).
Mitochondria distribution is a process mediated by a network of microtubules, and requires a high ATP level supplied through mitochondrial metabolism (Ge et al., 2012). Mitochondria modulate the amplitude and kinetics of local and bulk cytoplasmic Ca2+ changes; in addition, they are dependent on Ca2+ signals for their own functionality, especially their capacity to produce ATP. mCa2+ has been found to be an important component controlling the rate of ATP production, and it controls the occurrence of MPT, which plays a role in mitochondrial control of apoptosis (Zhao et al., 2015). This study therefore aimed to determine the optimum time of oocyte in vitro maturation and its correlations with apoptosis.
Materials and methods
This research received ethical approval with permit number 1.KEH.35.03.2021 by the Animal Ethics Committee of the Faculty of Veterinary Medicine, Universitas Airlangga.
The study materials included the following: Saline solution (0.9%) Otsu®.
The maturation medium used Minimum Essential Medium (MEM, GibcoBRL®), HEPES (Sigma-Aldrich®) stored at 4-5°C with 7.2 pH, Bovine Serum Albumin (BSA), Sodium bicarbonate (Sigma-Aldrich®, Steinheim, Germany), Kanamycin sulfate (Bioplus), Pregnant Mare Serum Gonadotropin (PMSG) (PG600®, Canada), Hormone Chorionic Gonadotropin (hCG) (PG600®, Canada).
Hyaluronidase (HYASE-10x™, Vitrolife®) for denudation, and apoptosis expression examination with immunocytochemistry (TACS-XL® DAB In Situ Apoptosis Detection Kit).
Collection and processing of ovaries
The ovaries were transported from slaughterhouses to the laboratory using a thermos. They were placed into a glass beaker containing 0.9% saline solution (0.9%) with penicillin G (75 μg/mL) and streptomycin sulfate (50 μg/mL) at 37°C. After trimming the surrounding tissues, ovaries were washed again with saline solution.
Collection of cumulus oocyte complexes
Cumulus oocyte complexes (COC) were aspirated in the follicles with a diameter of 2–6 mm using a 10 mL disposable syringe with an 18-G needle containing Phosphate Buffered Saline (PBS) medium. All aspirations were put into a Petri dish for evaluation under 40× magnification on an inverted microscope.
After evaluation, COCs containing healthy oocytes were selected based on their morphological appearance of a uniformly granulated cytoplasm surrounded by a multilayer of compact cumulus cells least three layers thick, and washed with PBS 3 times.
In vitro maturation of COCs
The maturation medium was prepared with Minimum Essential Medium (MEM) supplemented with 3% bovine serum albumin (BSA), 0.15 IU/mL Pregnant Mare Serum Gonadotropin (PMSG) and 15 IU/mL human chorionic gonadotropin (hCG).
COCs were placed in 50 µL maturation droplets of maturation medium under paraffin oil, drops were made on a 35 mm disposable Petri dish (each drop contained ±3–4 oocytes). They were cultured in an incubator with environmental conditions of 5% CO2, temperature of 37.5°C, and maximum humidity level (95–99%) for 18 hours, 22 hours or 24 hours.
Oocyte maturation of Kacang goat
After 18 h, 22 h or 24 h of culture, observations were made to see the level of oocyte maturity using a Nikon Diaphot 300 microscope at 100x and 400x magnification. To observe Polar body I, a denudation was performed to remove cumulus cells. Chemical denudation usually uses the enzyme hyaluronidase (HYASE-10x™, Vitrolife®), and the oocyte is immersed in a medium containing the hyaluronidase enzyme and left for 30 seconds then transferred to the culture medium. The maturity level of oocytes is measured by the number of oocytes that have undergone first polar body (PBI) release as a benchmark for oocyte maturation divided by the total number of oocytes multiplied by 100%.
Examination of apoptosis using immunocytochemistry
Oocytes were placed on a glass object coated with poly-L-lysine and then covered with a cover glass. Fixation of oocytes was conducted by putting the glass object into a container with 100% acetic acid (glacial) and absolute ethanol at a ratio of 1:3 for a minimum of 24 h before immunocytochemical colouring was performed. Slides were then deparaffinized in xylol 1, xylol 2, ethanol absolute 1, ethanol absolute 2, 90% ethanol, 80% ethanol, 70% ethanol, 30% ethanol, each for 5 minutes, then slides were washed in distilled water before staining with PBS for 5 minutes. Slides were covered with 50 µL proteinase K for 15–30 minutes or with 50 µL cytonin for 30 minutes. Then slides were washed with deionized water every 2 minutes before being dropped into quenching solution for 5 minutes and then washed with PBS for 1 minute. Slides were stained with 1xTDT labelling buffer for 5 minutes and then dropped into 50 µL Labelling reaction mix. This was followed by incubation at 37˚C in a damp place for 60 minutes. The sample was dripped with 1xTDT stop buffer for 5 minutes and then washed with sterile distilled water. After covering the slides with SA-HRP solution, they were incubated at 37˚C in a damp place for 10 minutes. This was followed by rinsing 2x with PBS for 2 minutes before dropping into DAB Solution for 2–7 minutes, and then rinsing 2x with sterile distilled water for 2 minutes. Slides were dropped into methyl green for 30 seconds to 5 minutes, and then soaked in a mixture 2x distilled water, 95% ethanol and absolute ethanol. Finally, slides were mounted using entellan.
Observation of apoptosis expression
Apoptosis expressions were observed using an CX41 microscope (Olympus, Japan). The results of each expression were assessed semi-quantitatively according to the modified Remmele method (Nowak et al., 2007). The index Remmele scale was the result of multiplying the percentage score of immunoreactive cells with the colour intensity scores produced on the cell, as shown in Table 1.
All data of oocyte maturity level and apoptosis were subjected to one-way ANOVA followed by Duncan test (IBM SPSS Statistics 25), and differences at P<0.05 were considered statistically significant.
Oocyte maturity level
The cumulus oocytes complexes that were successfully collected amounted to 25 oocytes for each group meeting the requirement for in vitro maturation in a different time period. The level of oocyte maturity obtained from maturation differed among treatment groups 1, 2 and 3. The number of mature oocytes marked by the formation of Polar Body I in treatment group 2 was higher than both groups 1 and 3 (Table 2).
According to Table 2, oocytes were able to experience in vitro maturation at 22 hours with a percentage of 77%. The mature oocytes are marked by the formation of Polar body I.
Apoptosis examination using immunocytochemistry
Apoptosis of oocytes in Kacang goat following in vitro maturation was expressed by brown colouration on both structures that indicated the bond of antigen and antibody as visualized by chromogenic change. According to Table 2, group 1 (T1) differed significantly from T2 (P<0.05), which also differed significantly from T3 (P<0.05). The expression of apoptosis in T2 (2.4 ± 0.89) was the lowest compared to the groups T1 (5.4 ± 2.61) and T3 (9.0 ± 2.21).
The result showed that the highest level of oocyte maturity was found in treatment group 2, after in vitro maturation of 22 hours (mean 77.14). This result was significant different (P<0.05) from treatment group 1 (45.71%), though group 3 (62.86%) did not significantly differ from groups 1 or 2. The release of first polar body (PBI) has been regarded as the benchmark of oocyte maturation.
However, our results indicate that spindle assembly may be very different in these seemingly matured oocytes and is closely related to the timing of in vitro maturation (Yu et al., 2011). Adequate extension of IVM time can promote the necessary process of oocyte maturation, increase the rate of nuclear maturation in immature oocytes, and significantly improve in vitro the developmental potential of oocytes and the rate of high-quality embryos (La et al., 2019). Souza-Fabjan et al. (2014) reported that 22 hours is the optimal time of in vitro maturation. On other hand, Maximo et al. (2012) reported that 18 h was sufficient for the oocyte to complete maturation.
They also suggested that degeneration signs may be due to the excessive time in IVM (24 h), which may disrupt the oocyte structures to induce apoptosis.
In addition to maturation data, oocytes were examined for apoptosis after in vitro maturation. The results on apoptosis are presented in Table 2 and Figure 1.
Brown colouration indicates an oocyte that experienced apoptosis, which would influence the nutritional supply from the cumulus to the oocyte. This led to the inability for the oocyte to achieve perfect maturity. Whereas, oocytes that did not experience apoptosis were expressed by staining counter colour that is methyl green. The oocytes did not experience apoptosis, enabling the oocyte to achieve the optimum time maturity in the metaphase II stage.
Based on the research, the lowest expression of apoptosis in treatment group T2 was significantly different from groups T1 and T3. Many factors influenced apoptosis to take place and accelerate the process. Excessive culture time leads to the aging of oocytes and increased level of associated genetic risks (La et al., 2019). A recent study suggested that a major cause for impaired developmental competence of aging oocytes is the induction of oxidative stress, which triggers many cascades that influence oocyte quality, such as apoptosis (Zhang et al., 2019). Oxidative stress coulbe due to cytochrome binding to apoptotic protease-activating factor-1 (APAF-1) released from the mitochondria; then procaspase-9 can activate caspase-9.43.
Caspase-9, which serves as an apoptosis initiator, is dimerized, triggering feedback by inhibiting BCL-2 release and then binding to procaspase-3 to activate caspase-3 (Safitri et al., 2023). Caspase3, which acts as an executor, may facilitate endonuclease activation and cytoplasmic proteases that may lead to nuclear DNA fragmentation and degradation of cytosol protein.
The final result in the fragmentation process is the formation of apoptotic bodies containing intracellular organelles, and the expression of phosphatidylserine that trigger phagocytosis (Samik and Safitri, 2017).
The authors would like to thank the Faculty of Veterinary Medicine, Universitas Airlangga, for providing the facilities for this research. The author hopes that the manuscript can be useful for further research on IVM or IVEP.
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Učinci trajanja postupka in vitro sazrijevanja na razinu sazrijevanja i apoptozu u kacang koza
Milla NURSADIDA, Widjiati WIDJIATI, Imam MUSTOFA, Erma SAFITRI, Suherni SUSILOWATI, Tri WAHYU SUPRAYOGI, Department of Veterinary Science Faculty of Veterinary Medicine Universitas Airlangga, Indonesia
Uspjeh in vitro oplodnje i kulture embrija ovisi o uspjehu in vitro sazrijevanja. Međutim, standard uvjeta kulture obično povećava reaktivne kisikove spojeve (ROS), za što se sumnja da je jedan od glavnih uzroka smanjenog razvoja embrija. Dobro je poznato da razine ROS veće od fizioloških razina izazivaju apoptozu granuloznih stanica i time smanjuju prijenos nutrijenata i faktora preživljavanja na oocite, što dovodi do apoptoze. Ovo istraživanje imalo je za cilj otkriti optimalno vrijeme sazrijevanja oocita i njegov odnos s DNK fragmentacijom. Prikupljeni su jajnici iz klaonice i aspirirani su folikuli. Kompleksi oocita-cumulus (COCs) podijeljeni su u skupine i preneseni u medij za sazrijevanje gdje su držani 18 sati (P1), 22 sata (P2) i 24 sata (P3) za procjenu postotka sazrijevanja. Zreli oociti su okarakterizirani putem oocita koji su dosegnuli MII fazu. Zreli oociti obojani su TUNEL metodom. Rezultati su pokazali da je postotak zrelosti koju su dosegli oociti kacang koze nakon 18 sati bio 46 %, nakon 22 h 77 % i nakon 24 h 63 %. Međutim, rezultati su pokazali da se ekspresija DNK fragmentacije u P2 (2,4 ± 0,89) značajno razlikovala od P1 (5,4 ± 2,61) i P3 (9,0 ± 2,12).
Zaključno, optimalno vrijeme in vitro sazrijevanja oocita kacang koze je 22 sata.
Ključne riječi: in vitro sazrijevanje (IVM), DNK fragmentacija, optimalno vrijeme, kacang koza, proizvodnja hrane