Effect of ferulic acid supplementation on the developmental competence of porcine embryos during in vitro maturation.

The value of laboratory and genetically-modified pigs is becoming increasingly clear; however, their in vitro development remains inefficient. Trans-ferulic acid (trans-FA) is an aromatic compound that is abundant in plant cell walls, and which exhibits antioxidant effects in vitro. Trans-FA is known to improve sperm viability and motility; however, its effects on porcine oocytes are unknown. Our aim was to investigate the effects of trans-FA supplementation during in vitro maturation on the meiotic and developmental competence of porcine oocytes. Oocytes were matured either without (control) or with trans-FA (10, 100 and 1,000 µM), fertilized, and cultured in vitro for 7 days. The maturation rate of oocytes cultured with 10 µM trans-FA (81.6%) was significantly higher than that of controls (65.0%; P<0.05). The fertilization rate of oocytes matured with 10 µM trans-FA (57.4%) was also significantly higher than that of controls (32.7%) and oocytes cultured with other concentrations (33.1% and 22.7% for 100 and 1,000 µM, respectively; P<0.05). Moreover, the blastocyst formation rate of oocytes matured with 10 µM trans-FA (6.9%) was significantly higher than that of controls (2.3%; P<0.05). Our results suggest that in vitro maturation with 10 µM trans-FA is beneficial for the in vitro production of porcine embryos and has the potential to improve production system.

Recently, the value of pigs as laboratory animals has become widely recognized. Anatomically, and physiologically, pigs have close similarity to humans. Moreover, the genetic modification of these animals is expected to facilitate the generation of excellent human disease models, the production of suitable organs for xenotransplantation, and research on human regenerative medicine. Genetically modified pigs are generated through the molecular manipulation of embryos with subsequent implantation into surrogate pigs. In these animals, gene modification is normally performed using in vitro matured oocytes or subsequently fertilized zygotes. Therefore, in vitro matured oocytes are a critical resource for the generation of genetically modified pigs. To date, several researchers have tried to improve this in vitro production (IVP) system as well as the quality of in vitro matured oocytes, including their developmental competence after in vitro fertilization (IVF) and culture (IVC) [11, 17, 18]. However, the percentage of development to the blastocyst stage of in vitro produced embryos is still lower compared to that of in vivo produced ones [13, 26].

The high oxygen concentration and lack of antioxidant protection associated with in vitro conditions result in the increased generation of reactive oxygen species (ROS), and in turn, increased oxidative stress for the oocytes [2, 3]. Antioxidant supplementation during oocyte/embryo culture has been reported to reduce the effects of ROS, resulting in the protection of oocytes/embryos against damage caused by oxidative stress and improved IVP systems to generate high-quality porcine embryos [29]. In previous studies, the use of antioxidant such as flavonoids (quercetin and taxifolin) [16], melatonin [8, 10], selenium [30], vitamin E [30], resveratrol [21], chlorogenic acid (CGA) and caffeic acid [24] during in vitro maturation (IVM) has improved the production system for porcine embryos. Trans-ferulic acid (trans-FA) is an aromatic compound that is abundant in plant cell walls [7, 28]. Trans-FA removes ROS and exhibits an antioxidant effect in vitro [25, 28]. In humans, it was previously shown that supplementing the culture medium with trans-FA improves sperm viability and motility [35]. However, the effects of this compound on IVM, IVF, and IVC for porcine oocytes remains unknown. Therefore, in the present study, we investigated the effects of trans-FA supplementation during IVM on the meiotic and developmental competence of porcine oocytes.

MATERIALS AND METHODS

In vitro maturation and assessment of meiotic competence

Porcine ovaries were obtained from approximately 6-month-old gilts at a local slaughterhouse and were transported within 1 hr to the laboratory in physiological saline at 30°C. Ovaries were washed three times with modified phosphate-buffered saline (m-PBS; Nihonzenyaku, Fukushima, Japan) supplemented with 100 IU/ml penicillin G potassium (Meiji, Tokyo, Japan) and 0.1 mg/ml streptomycin sulfate (Meiji). The follicles of the ovarian surface were sliced using a surgical blade on a sterilized dish, and the cumulus-oocyte complexes (COCs) were collected under a stereomicroscope. Only COCs with a uniformly dark-pigmented ooplasm and intact cumulus cell masses were collected. Approximately 50 COCs were then cultured in 500 µl of maturation medium, consisting of tissue culture medium 199 with Earle’s salts (TCM 199; Invitrogen Co., Carlsbad, CA, U.S.A.), supplemented with 10% (v/v) porcine follicular fluid, 50 µM sodium pyruvate (Sigma-Aldrich, St. Louis, MO, U.S.A.), 2 mg/ml D-sorbitol (Wako Pure Chemical Industries Ltd., Osaka, Japan), 10 IU/ml equine chorionic gonadotropin (Kyoritu Seiyaku, Tokyo, Japan), 10 IU/ml human chorionic gonadotropin (Kyoritu Seiyaku), and 50 µg/ml gentamicin (Sigma-Aldrich), covered with mineral oil (Sigma-Aldrich) for 22 hr in 4-well dishes (Nunc A/S, Roskilde, Denmark). Subsequently, the COCs were transferred into maturation medium without hormone supplementation and cultured for an additional 22 hr. The incubation of COCs was conducted at 39°C in a humidified incubator containing 5% CO2 in air.

To assess the meiotic status of oocytes following IVM, some oocytes were denuded, fixed, and permeabilized in Dulbecco’s PBS (DPBS; Invitrogen) supplemented with 3.7% (w/v) paraformaldehyde and 1% (v/v) triton X-100 (Sigma-Aldrich) at 25°C for 15 min. Permeabilized oocytes were then placed on glass slides and stained with 1.9 mM bisbenzimide (Hoechst 33342; Sigma-Aldrich), before being mounted with coverslips. After overnight incubation at 4°C, the oocytes were examined by fluorescence microscopy. Based on their chromatin configuration, they were classified as ‘germinal vesicle,’ ‘condensed chromatin,’ ‘metaphase I,’ or ‘metaphase II’. Oocytes with the diffusely stained cytoplasmic characteristics of nonviable cells and those in which chromatin was unidentifiable or not visible were classified as ‘degenerated’.

IVF and assessment of fertilization

IVF was performed according to methods described previously [24] with minor modifications. Frozen-thawed ejaculated spermatozoa were transferred into 5 ml of fertilization medium (PFM; Research Institute for the Functional Peptides Co., Yamagata, Japan) and washed by centrifugation at 500 × g for 5 min. The pelleted spermatozoa were resuspended in fertilization medium and adjusted to 1 × 106 cells/ml. Next, approximately 50 COCs were transferred to 500 µl of sperm-containing fertilization medium covered with mineral oil in 4-well dishes and co-incubated for 5 hr at 39°C with 5% CO2 and 5% O2. After the co-incubation, the inseminated zygotes were denuded from the cumulus cells and the attached spermatozoa by mechanical pipetting.

To assess fertilization of the oocytes, some denuded zygotes were mounted on glass slides and fixed with acetic acid:ethanol (1:3 v/v) for 72 hr. The fixed zygotes were stained with acetic orcein (1% orcein in 45% acetic acid) and examined by phase contrast microscopy. Oocytes containing both female and male pronuclei were considered fertilized and were categorized as monospermic or polyspermic according to the number of swollen sperm heads and pronuclei in the cytoplasm.

IVC and assessment of blastocyst quality

The remaining denuded zygotes were subsequently transferred to PZM-5 (Research Institute for the Functional Peptides Co.). Approximately 50 zygotes were cultured continuously in 500 µl of PZM-5 covered with mineral oil in 4-well dishes. After culturing the embryos for 3 days, they were subsequently incubated in 500 µl of porcine blastocyst medium (PBM; Research Institute for the Functional Peptides Co.) covered with mineral oil for 4 days in 4-well dishes. The incubation of zygotes and embryos was conducted at 39°C in a humidified incubator containing 5% CO2, 5% O2 and 90% N2. To evaluate the development stage of fertilized oocytes, all embryos were fixed on day 7 (day 0=insemination) and were stained with Hoechst 33342 to assess the quality of embryos through cell counting.

Experimental design

To evaluate the effects of trans-FA supplementation during IVM culture on the in vitro maturation, fertilization, and development of porcine oocytes, COCs were cultured in maturation medium supplemented with 10, 100 and 1,000 µM trans-FA (Sigma-Aldrich). As a control, COCs were cultured in maturation medium without trans-FA. After maturation culture for 44 hr, COCs were fertilized in vitro and cultured continuously as described.

Statistical analysis

Experiments were repeated five times for oocytes matured with trans-FA. All percentage data were subjected to arcsine transformation before performing an analysis of variance (ANOVA). The transformed data were tested by ANOVA, followed by a Fisher’s protected least significant difference (LSD) test, using StatView software (Abacus Concepts, Berkeley, CA, U.S.A.). Differences with a probability value (P) of 0.05 or less were considered statistically significant.

RESULTS

As shown in Table 1, the maturation rate of oocytes cultured with 10 µM trans-FA (81.6%) was significantly higher (P<0.05) than that of controls (65.0%). Moreover, the fertilization rate of oocytes cultured with 10 µM trans-FA (57.4%) during IVM was significantly higher (P<0.05) than that of controls (32.7%) and oocytes cultured with 100 µM (33.1%) and 1,000 µM (22.7%) trans-FA. However, trans-FA treatment had no effect on monospermy rates among the groups. The cleavage rate of oocytes matured with 10 µM trans-FA (86.3%) was significantly increased compared to that with other concentrations of trans-FA (72.4 and 75.2% for 100 and 1,000 µM, respectively; P<0.05). Moreover, the blastocyst formation rate of oocytes matured with 10 µM trans-FA (6.9%) was significantly higher (P<0.05) than that of controls (2.3%).

Table 1.

Effects of trans-ferulic acid (trans-FA) supplementation during in vitro maturation culture on the maturation, fertilization and development of porcine oocytes*

Concentration of trans-FA (µM)	No. of examined oocytes	No. (%)** of matured oocytes	No. of examined oocytes	No. of oocytes		No. of examined embryos	No. of embryos		No. of cells in blastocyst
				Fertilized (%)	Monospermy (%)***		Cleaved (%)	Developed to blastocysts (%)
0	72	49 (65.0 ± 3.6)a)	64	22 (32.7 ± 4.1)a)	18 (82.7 ± 8.4)	181	151 (83.1 ± 3.3)a,b)	4 (2.3 ± 0.6)a)	45.9 ± 10.6
10	72	60 (81.6 ± 5.6)b)	63	35 (57.4 ± 11.5)b)	26 (80.5 ± 10.7)	189	164 (86.3 ± 3.3)a)	14 (6.9 ± 2.1)b)	36.3 ± 4.1
100	70	46 (66.2 ± 8.1)a,b)	66	19 (33.1 ± 7.9)a)	13 (66.3 ± 11.6)	193	141 (72.4 ± 5.2)b)	9 (4.4 ± 1.5)a,b)	36.9 ± 4.1
1,000	69	48 (67.3 ± 5.9)a,b)	65	15 (22.7 ± 10.5)a)	13 (91.4 ± 5.1)	158	119 (75.2 ± 3.3)b)	5 (3.1 ± 0.9)a,b)	35.1 ± 3.3

*Five replicated trials were carried out. **Percentages are expressed as mean ± SEM. ***The monospermic fertilization rate was defined as a ratio of the number of monospermic oocytes and the total number of fertilized oocytes. a, b) Values with different superscripts in the same column differ significantly (P<0.05).

DISCUSSION

In the present study, we confirmed the potential of trans-FA as an additive agent to maturation medium. We found that supplementation with 10 µM trans-FA significantly improved the rates of maturation, fertilization, and blastocyst formation in oocytes.

To date, many improvements have been made to in vitro culture systems to produce high-quality IVF embryos; however, the developmental competence of in vitro embryos remains insufficient compared to that with in vivo embryos [9, 18, 19]. One factor for the success of IVP has been suggested to be the oxidative conditions of the environment surrounding the oocytes and embryos [20]. The oxygen concentration in atmospheric air is approximately three-fold greater than that found under the lumen of the female reproductive tract [5, 23, 27]. Oxidative stress via ROS is a threat to oocytes and embryos in vitro, which have been removed from their natural habitat and place into a harsh environment that lacks maternal antioxidant factors. Oxidative stress during oocyte maturation, which gives rise to mitochondrial damage [31], DNA damage and apoptosis [29], can affect subsequent fertilization [4] and impair embryo development [20]. One study using human embryos demonstrated that the culture media itself can be a source of ROS [22].

Oocytes are protected from oxidative stress in vivo by external and internal protection systems [15]. External protection is mainly composed of non-enzymatic antioxidants such as hypotaurine, taurine, and ascorbic acid, which are contained in follicular and tubal fluids. Conversely, internal protection is mainly composed of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and gamma-glutamylcysteine synthetase. In vivo, an efficient reduction-oxidation reaction (redox) system can ensure an ideal environment for embryonic development by maintaining a balance between the production of ROS and antioxidant enzymes [1, 15]. However, in vitro conditions for oocyte culture lack external non-enzymatic antioxidants protection, which leads to increase ROS [15]. Therefore, antioxidant supplementation, as external protection against oxidative stress, could be an effective strategy for in vitro oocyte culture.

In our previous study, we suggested that CGA or caffeic acid supplementation during IVM are beneficial for oocyte maturation, fertilization, and the development to blastocysts [24]. CGA is a quinic acid conjugate of caffeic acid [14] and caffeic acid is the precursor to ferulic acid [6]. Trans-FA has a similar structure to CGA and caffeic acid, and therefore it is possible that trans-FA is also beneficial for the IVP of porcine embryos. In the present study, trans-FA supplementation significantly enhanced the nuclear maturation of oocytes and accelerated fertilization. Furthermore, embryonic development following IVF was significantly increased, compared to that in the control group. During porcine IVP, polyspermic penetration is a major obstacle for the production of normal and high-quality embryos [11, 12]. In this study, supplementation with 10 µM trans-FA significantly increased the fertilization rate compared to that in the control group; however, the monospermy rate was comparable to that in the control group, which indicated that trans-FA supplementation during IVM increased the number of normal fertilized embryos. These results suggested that this treatment enhances the nuclear maturation and normal fertilization of porcine oocytes, resulting in a higher blastocyst formation. However, the blastocyst formation rate was lower in all groups including control group compared to that of our previous study [14]. In this study, we did not add cysteine, well known as one of the effective additives to improve developmental competence of oocytes during IVM [32,33,34], to IVM medium to evaluate the effects of trans-FA supplementation clearly. We guess that the lack of supplementation of IVM medium with cysteine reduced embryonic development.

In conclusion, trans-FA is an effective additive that improves the maturation, fertilization, and developmental competence of porcine oocytes. It appears that trans-FA supplementation in maturation medium could be used in porcine IVP systems, and thus might facilitate further developments in biotechnology.