Effect of rapamycin treatment during post-activation and/or in vitro culture on embryonic development after parthenogenesis and in vitro fertilization in pigs
Contents
This study investigated the effects of early induction of autophagy on embryonic development in pigs. For this, oocytes or embryos were treated with an autophagy inducer, rapamycin (RP), during post-activation (Pa), in vitro fertilization (IVF) and/or in vitro culture (IVC). When parthenogenesis (PA) embryos were untreated (control) or treated with various concentrations of RP for 4 hr during Pa, 100 nm RP showed a higher blastocyst formation (48.8 ± 2.7%) than the control (34.6 ± 3.0%). When PA embryos were treated during the first 24 hr of IVC, blastocyst formation was increased (p < .05) by 1 and 10 nm RP (61.9 ± 3.0 and 59.6 ± 3.0%, respectively) compared to the control (43.2 ± 1.8%) and 100 nm RP (47.8 ± 3.2%), with a higher embryo cleavage in response to 10 nm RP (87.3 ± 2.4%) than the control (74.1 ± 3.2%). RP treatment during IVC and Pa + IVC showed increased blastocyst formation (44.7 ± 2.5 and 44.1 ± 2.0%, respectively) compared to the control (33.2 ± 2.0%). In addition, RP treatment during Pa and/or IVC increased glutathione content and inversely reduced reactive oxygen species. In IVF, RP treatment for 6 hr during IVF significantly increased embryonic development (34.0 ± 2.6%) compared to the control (24.8 ± 1.6%), but treatment during IVC for 24 hr with RP did not (23.0 ± 3.8%). Autophagy was significantly increased in PA oocytes by the RP treatment during Pa but not altered by the treatment during the first 24 hr of IVC. Overall, RP treatment positively regulated the pre-implantation development of pig embryos, probably by regulating cellular redox state and stimulating autophagy.
1 INTRODUCTION
Pigs are useful as an animal model for biomedical research (Rogers et al., 2008) because of their physiological similarity to humans (Abeydeera, 2002; Betthauser et al., 2000). In vitro production (IVP) of pig embryos basically depends on various vital procedures such as in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC), which are widely applied to produce embryos or animals for the livestock industry as well as biomedical research for transgenesis and xenotransplantation (Kawamura et al., 2012; Lai et al., 2002). However, the poor quality of IVP embryos compared to in vivo-grown embryos is a major obstacle to the production of embryos with high developmental competence (Kim, Ahn, Kim, & Shim, 2011; Lee et al., 2016). Many investigators have attempted to improve the quality of IVP embryos by optimizing an IVM and IVC systems for pig oocytes by adding a variety of substances to culture medium (Krisher, 2004; Lee et al., 2016).
Autophagy is important for various physiological processes such as adaptation to starvation, quality control of cytoplasmic constituents and recovery of cells from intracellular pathogens (Levine & Klionsky, 2004; Mizushima & Komatsu, 2011; Othman, Kaur, Mutee, Muhammad, & Tan, 2009). Autophagy or autophagocytosis is a physiological process that removes unwanted or degraded cellular proteins and components (Ferraro & Cecconi, 2007). Autophagy helps reconstruction within a cell population, such as embryonic cavitation and patterning. This process dynamically governs intracellular degradation, which is essential for cellular processes such as amino acid production during starvation and intracellular quality control (Kawamura et al., 2012; Qu et al., 2007; Yamamoto, Mizushima, & Tsukamoto, 2014). Mammalian target of rapamycin (mTOR), a conserved Ser/Thr protein kinase, inhibits the autophagy process (Foster & Fingar, 2010). Conversely, mTOR inhibitors such as RP can activate autophagy function in mammalian cells (Sully et al., 2013). A recent study showed that autophagy has positive effects on embryonic generation in mammals. In mice, knockout of autophagy genes halted embryonic development at the 4- to 8-cell stage, resulting in death of all embryos (Tsukamoto, Kuma, Murakami, et al., 2008). In a bovine model, autophagy induced transiently using a chemical increased the pre-implantation development of embryos (Song et al., 2012).
In pig embryos, autophagy degrades maternal mRNA and regulates apoptosis in pigs, playing an important role in early embryonic development (Xu et al., 2012). A previous study in mice reported that autophagy plays an important role in pre-implantation development of embryos (Tsukamoto, Kuma, & Mizushima, 2008). Moreover, unfertilized oocytes showed a low activity of autophagy that quickly increased within 4 hr of fertilization (Tsukamoto, Kuma, Murakami, et al., 2008). Thus, the porcine IVC method must be further improved by modulation of the developmental events governing embryo development. Previously, RP was used in IVM for pig oocyte maturation and found a positive influence on embryonic development (Lee et al., 2015; Song et al., 2014). However, the effects of autophagy inducers during the post-activation (Pa) period and the early stages of embryonic development after PA and IVF have not been clarified in pigs. We investigated whether this high level of autophagy induction would be important to exchanging the maternal cytoplasmic contents into zygotic materials and for improving quality, viability and proportion of embryos in assisted reproductive technology.
2 MATERIALS AND METHODS
2.1 Experimental design
Experiments 1 and 2 were performed to determine the optimal concentration of RP for embryonic development. In Experiment 1, after electrical activation (EA) of oocytes, 0 (control), 1, 10 or 100 nm RP were added into IVC medium for 4 hr during the Pa treatment because physiologically, autophagy induction starts immediately after fertilization (Tsukamoto, Kuma, Murakami, et al., 2008). After treatment, parthenogenesis (PA) embryos were cultured in IVC medium for 7 days. In Experiment 2, PA embryos were cultured for 24 hr in IVC medium supplemented with 0 (control), 1, 10 and 100 nm RP and then subsequently cultured without RP for another 6 days. Based on the results of Experiments 1 and 2, we selected 10 nm RP for further experiments because both the embryo cleavage and blastocyst formation were significantly increased by this concentration. In Experiment 3, the effects of RP treatment during Pa and/or the first 24 hr of IVC on embryonic development were evaluated. Anti-oxidative substance such as glutathione (GSH) protects oocytes and embryos from the harmful action of reactive oxygen species (ROS), and thus, intracellular GSH content is frequently used as an indicator to assess the developmental competence of oocytes and embryos (Sakatani et al., 2007; You, Kim, Lim, & Lee, 2010). In Experiment 4, effect of autophagy induction on GSH and ROS contents was determined in two-cell PA embryos that were untreated or treated with RP during Pa and/or IVC. In Experiment 5, PA embryos were treated with RP during IVC for 1, 3, 5 and 7 days to determine optimal treatment period for embryonic development. RP treatment efficacy in embryonic development was evaluated by treating oocytes with RP during IVF for 6 hr and/or for the first 24 hr of IVC in Experiment 6. Finally, autophagy activities were evaluated before and after EA, after Pa and after 24 hr of IVC in PA oocytes that were treated or untreated with RP in Experiment 7.
The experimental procedures used in this study were approved by the Institutional Animal Care and Use Committee of Kangwon National University in accordance with the Guiding Principles for the Care and Use of Research Animals.
2.2 Culture media and reagents
All reagents and chemicals were bought from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. RP was dissolved in DMSO as previously described (Lee et al., 2015). IVM of oocytes was conducted in Medium-199 (M-199; Invitrogen, Grand Island, NY, USA) containing 0.6 mm cysteine, 10% (vol/vol) porcine follicular fluid, 10 ng/ml epidermal growth factor, 0.91 mm pyruvate, 75 μg/ml kanamycin and 1 μg/ml insulin. Porcine zygote medium (PZM)-3 supplemented with 2.77 mm myo-inositol, 10 μm β-mercaptoethanol and 0.34 mm trisodium citrate was used as the IVC medium for embryo culture (Lee et al., 2015).
2.3 IVM of oocytes
Pig ovaries were collected from a local slaughter house and transported to the laboratory in warm physiological saline within 1 hr. The cumulus–oocyte complexes (COCs) were collected from follicles 3–8 mm in diameter with using a 10-ml syringe with an 18-gauge needle. The COCs were then washed in HEPES-buffered Tyrode's medium containing 0.05% (wt/vol) polyvinyl alcohol (PVA) (TLH-PVA) three times, after which uniform ooplasms and multiple layers of compacted cumulus cells were cultured in 500 μl IVM medium with 10 IU/ml hCG (Intervet International BV, Boxmeer, Holland) and 80 μg/ml FSH (Antrin R-10; Kyoritsu Seiyaku, Tokyo, Japan) in a four-well multidish (Nunc, Roskilde, Denmark) at 39°C with 5% CO2 and maximum humidity. After 22 hr of IVM, the COCs were transferred in fresh hormone-free IVM media following washing three times in the same IVM medium and then cultured for an additional 22 hr for PA and IVF.
2.4 PA, IVF and embryo culture
After 44 hr of IVM, the cumulus cells of COCs were removed manually in the presence of 0.1% (wt/vol) hyaluronidase. For PA, oocytes having the first polar body (MII) were electrically activated in 280 mm mannitol solution supplemented with 0.05 mm MgCl2 and 0.1 mm CaCl2 by applying two pulses of 120V/mm direct current for 60 μs. The PA embryos were exposed to 5 μg/ml cytochalasin B and/or RP in 1, 10 or 100 nm in IVC medium for 4 hr after EA according to the experimental design. For IVF, extended liquid semen (Geumbo Genetics, Wonju, Korea) was purchased weekly and stored at 17°C before use. Semen was washed twice by centrifugation at 350 g for 3 min in Dulbecco's phosphate-buffered saline (D-PBS; Invitrogen) containing 0.1% (w/v) BSA. After washing, the final sperm pellet was resuspended in modified Tris-buffered medium containing 0.8% (w/v) BSA (Hong & Lee, 2007) to a concentration of 1.2 × 106 spermatozoa/ml. Then, 15–20 MII oocytes were co-cultured with sperm in 50 μl IVF droplets for 6 hr at 39°C under 5% CO2 and maximum humidity. The final sperm concentration for IVF was 1.2 × 105 spermatozoa/ml. After co-incubation, loosely attached sperm were dispersed by gentle pipetting and washed three times in IVC medium. PA and IVF embryos were placed into 30 μl IVC droplets, covered with mineral oil and cultured at 39°C in a humidified atmosphere of 90% N2, 5% O2 and 5% CO2 for 7 days. Cleavage and blastocyst formation were evaluated on Days 2 and 7, respectively. The day of PA and IVF was considered Day 0. The total cell count in blastocysts was conducted using Hoechst 33342 staining under an epifluorescence microscope.
2.5 Measurement of intra-embryo GSH and ROS content
After 24 hr of IVC, PA embryos were examined for intra-embryo GSH and ROS contents as previously described (You et al., 2010). Briefly, Cell-Tracker Blue 4-chloromethyl-6.8-difluoro-7-hydroxycoumarin (Invitrogen) and H2DCFDA (2′,7′-dichlorodihydro-fluorescein diacetate; Invitrogen) were used to detect GSH and ROS with blue fluorescence and green fluorescence, respectively. A group of 7–10 embryos from each treatment group were cultured for 30 min in TLH-PVA supplemented with 10 μm H2DCFDA and 10 μm Cell-Tracker in the dark. Embryos treated with Cell-Tracker were then incubated for 30 min with PZM-3 supplemented with 0.3% (wt/vol) BSA at 39°C in the dark. Following incubation, the embryos were washed with D-PBS containing 0.01% (wt/vol) PVA, placed into 2 μl droplets. Fluorescence was detected under an epifluorescence microscope (TE300; Nikon) with ultraviolet ray filters at 370 and 460 nm for GSH and ROS, respectively. The fluorescence intensities of oocytes were examined by the imagej software (version 1.46r; National Institutes of Health, Bethesda, MD, USA) and normalized against untreated control oocytes.
2.6 Measurement of autophagy activity in PA oocytes
Autophagy activity was determined in one-cell stage oocytes before EA, just after EA and after Pa treatment and also in two-cell stage embryos after 24 hr of IVC. The Cyto-ID® Green autophagy dye has been shown previously to specifically detect autophagy in live cells (Chan et al., 2012). Autophagy level was detected using the Cyto-ID® Green autophagy dye (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer's protocol. In brief, the Cyto-ID® Green autophagy dye solution was prepared by mixing 1 μl of the dye and 500 μl of 1× assay buffer. The oocytes were incubated in this dye for 30 min at 37°C in dark, followed by a wash with 1× assay buffer, transferred into 2 μl of 1× assay buffer and covered with mineral oil. Then, fluorescence signals were captured with a digital camera (DS-L3; Nikon) under an inverted epifluorescence microscope (TE300; Nikon) and processed with imagej software (version 1.46r; National Institutes of Health). The fluorescence intensity was normalized against that of oocytes not electrically activated or oocytes not treated with RP during Pa and IVC, respectively.
2.7 Statistical analysis
All data were analysed with the Statistical Analysis System (version 9.3; SAS Institute, Cary, NC, USA). Analysis using a general linear model procedure was conducted and followed by the least significant difference mean separation procedure when treatments differed at p < .05. The percentage data were subjected to arcsine transformation before analysis for homogeneity of variance. The results are reported as the means ± the standard error of the mean.
3 RESULTS
3.1 Effects of RP treatment during the Pa period on embryonic development after PA (Experiment 1)
When PA embryos were treated for 4 hr after activation with 0, 1, 10 and 100 nm RP, embryo cleavage was not influenced by RP treatments. Conversely, blastocyst development was significantly (p < .05) increased in 100 nm RP relative to the control and other treatment counterparts (34.6 ± 3.0, 44.6 ± 6.1, 46.6 ± 4.7 and 48.8 ± 2.7% for control, 1, 10 and 100 nm of RP, respectively [Table 1]). The average cell number of blastocysts did not differ among groups tested.
| Rapamycin treatment | No. of PA embryos cultured | Percentage of embryos developed to | No. of cells in blastocyst | |
|---|---|---|---|---|
| ≥2 cells | Blastocyst | |||
| Control | 185 | 86.0 ± 2.7 | 34.6 ± 3.0a | 34.0 ± 1.5 |
| 1 nm | 187 | 90.1 ± 2.7 | 44.6 ± 6.1ab | 38.0 ± 1.5 |
| 10 nm | 184 | 93.0 ± 2.4 | 46.1 ± 4.7ab | 37.6 ± 1.5 |
| 100 nm | 174 | 90.1 ± 1.7 | 48.8 ± 2.7b | 34.4 ± 1.3 |
- Six replicates. PA oocytes were treated for 4 hr after electrical activation. abWithin a column, treated values with different superscripts are different (p < .05).
3.2 Effects of RP treatment during the first 24 hr of IVC on embryonic development after PA (Experiment 2)
We tested the effects of artificial restoration of autophagy during the first 24 hr of IVC with various concentrations of RP because autophagocytosis generally is considered to occur at the two-cell stage of embryonic development. Embryo cleavage was significantly augmented by 10 nm RP compared to the control (87.3 ± 2.4% vs 74.1 ± 3.2%). A similar pattern of blastocyst formation was evident in embryos treated with 1 and 10 nm RP (61.9 ± 3.0 and 59.6 ± 3.0%, respectively) compared to the control (43.2 ± 1.8%) and 100 nm RP (47.8 ± 3.2%) groups. However, the mean cell number in blastocysts did not differ after treatment with various concentrations of RP (33.3–36.7 cells/blastocyst) (Table 2).
| Rapamycin treatment | No. of PA embryos cultured | Percentage of embryos developed to | No. of cells in blastocyst | |
|---|---|---|---|---|
| ≥2 cells | Blastocyst | |||
| Control | 146 | 74.1 ± 3.2a | 43.2 ± 1.8a | 35.2 ± 1.6 |
| 1 nm | 142 | 81.7 ± 4.5ab | 61.9 ± 3.0b | 36.7 ± 1.4 |
| 10 nm | 141 | 87.3 ± 2.4b | 59.6 ± 3.0b | 35.4 ± 1.4 |
| 100 nm | 140 | 82.1 ± 3.2ab | 47.8 ± 3.2a | 33.3 ± 1.3 |
- Four replicates. PA embryos were treated with rapamycin for 24 hr of in vitro culture. abWithin a column, values with different superscripts are different (p < .05).
3.3 Effects of RP treatment during Pa and/or IVC on embryonic development after PA (Experiment 3)
Embryos were cultured in the presence or absence of 10 nm RP during Pa and/or the first 24 hr of IVC. Blastocyst formation was significantly higher in the Pa + IVC (44.1 ± 2.0%) and IVC treatment (44.7 ± 2.5%) than the control (33.2 ± 2.0%). On the other hand, cleavage and mean cell number in blastocysts were not influenced by RP treatments (Table 3).
| Rapamycin treatment during | No. of PA embryos cultured | Percentage of embryos developed to | No. of cells in blastocyst | ||
|---|---|---|---|---|---|
| Pa (4 hr) | IVC (24 hr) | ≥2 cells | Blastocyst | ||
| No | No | 239 | 93.1 ± 1.3 | 33.2 ± 2.0a | 36.2 ± 2.0 |
| No | Yes | 231 | 93.2 ± 2.0 | 44.7 ± 2.5b | 34.4 ± 1.0 |
| Yes | No | 228 | 90.8 ± 2.1 | 40.4 ± 4.6ab | 34.8 ± 1.0 |
| Yes | Yes | 221 | 94.1 ± 2.3 | 44.1 ± 2.0b | 33.4 ± 1.1 |
- Four replicates. Embryos were treated with 10 nm rapamycin. abWithin a column, values with different superscripts are different (p < .05).
3.4 Effects of RP treatment during Pa and/or IVC on GSH and ROS contents in PA embryos (Experiment 4)
Effect of RP treatment during Pa and/or IVC on GSH and ROS contents was evaluated. The results revealed RP treatments during Pa and/or IVC significantly (p < .05) increased GSH contents and decreased ROS levels in embryos compared to the control (Table 4).
| Rapamycin treatment during | No. of embryos examined for GSH | GSH content (pixels/embryo) | No. of embryos examined for ROS | ROS level (pixels/embryo) | |
|---|---|---|---|---|---|
| Pa (4 hr) | IVC (24 hr) | ||||
| No | No | 31 | 1.00 ± 0.02a | 38 | 1.00 ± 0.07a |
| No | Yes | 31 | 1.12 ± 0.01b | 38 | 0.81 ± 0.05b |
| Yes | No | 31 | 1.15 ± 0.02b | 38 | 0.80 ± 0.04b |
| Yes | Yes | 31 | 1.14 ± 0.01b | 38 | 0.78 ± 0.05b |
- Embryos were treated with 10 nm rapamycin. GSH and ROS levels were determined in two-cell stage embryos after 24 hr of IVC. abWithin a column, values with different superscripts are different (p < .05).
3.5 In vitro development of PA Embryos after treatment with RP during various periods of IVC (Experiment 5)
PA embryos were exposed to 10 nm RP for various periods of IVC. The results revealed that blastocyst formation was significantly higher (p < .05) in 1- and 3-day treatments (57.0 ± 5.3 and 54.7 ± 2.2%, respectively) than the control (41.2 ± 1.7%). However, there was no significant effect of RP on cleavage and cell number in blastocysts among groups (Table 5).
| Rapamycin treatment during IVC (day) | No. of PA embryos cultured | Percentage of embryos developed to | No. of cells in blastocyst | |
|---|---|---|---|---|
| ≥2 cells | Blastocyst | |||
| Control | 145 | 80.6 ± 1.2 | 41.2 ± 1.7a | 40.5 ± 1.9 |
| 1 | 147 | 87.0 ± 3.2 | 57.0 ± 5.3b | 41.6 ± 1.8 |
| 3 | 145 | 88.5 ± 2.3 | 54.7 ± 2.2b | 39.6 ± 1.3 |
| 5 | 144 | 84.4 ± 4.1 | 49.1 ± 3.2ab | 38.5 ± 2.0 |
| 7 | 145 | 85.6 ± 3.8 | 52.2 ± 2.3ab | 41.0 ± 1.8 |
- Five replicates. PA embryos were treated with 10 nm rapamycin for various periods of IVC. abWithin a column, values with different superscripts are different (p < .05).
3.6 Effects of RP treatment during IVF and/or IVC on in vitro development of IVF embryos (Experiment 6)
The effects of autophagy induction were evaluated in IVF embryos by treating oocytes with RP during IVF and/or the first day of IVC. RP treatment for 6 hr during IVF significantly improved embryonic development to the blastocyst stage (34.0 ± 2.6%) compared to the control (24.8 ± 1.6%). Extended exposure to RP during IVF and IVC showed detrimental effects on blastocyst formation (16.1 ± 3.2%) compared to the untreated control (Table 6). Embryo cleavage and mean cell number per blastocyst were not influenced by RP.
| Rapamycin treatment during | No. of IVF embryos cultured | Percentage of embryos developed to | No. of cells in blastocyst | ||
|---|---|---|---|---|---|
| IVF (6 hr) | IVC (24 hr) | ≥2 cells | Blastocyst | ||
| No | No | 148 | 53.9 ± 3.3 | 24.8 ± 1.6a | 34.6 ± 1.4 |
| No | Yes | 147 | 61.4 ± 3.7 | 23.0 ± 3.8ac | 37.3 ± 2.6 |
| Yes | No | 148 | 58.8 ± 5.8 | 34.0 ± 2.6b | 34.0 ± 1.8 |
| Yes | Yes | 148 | 53.4 ± 5.5 | 16.1 ± 3.2c | 40.8 ± 3.0 |
- Five replicates. Embryos were treated with 10 nm rapamycin. a–cWithin a column, values with different superscripts are different (p < .05).
3.7 Autophagy activity in RP-treated oocytes at various stages of EA, Pa and IVC (Experiment 7)
The autophagy activity was significantly (p < .01) increased by EA (1.10 ± 0.03 pixel/oocyte) compared to that of not activated oocytes (1.00 ± 0.03 pixel/oocyte). In addition, treatment with 10 and 100 nm RP during Pa period significantly increased (p < .01) the autophagy activity compared to no treatment (1.14 ± 0.03, 1.24 ± 0.03 and 1.28 ± 0.02 pixels/oocyte for no treatment, 10 nm and 100 nm RP, respectively). However, autophagy activities at 24 hr of IVC (0.96 ± 0.02–1.00 ± 0.03 pixels/oocyte) were not altered by the RP treatment during Pa and/or IVC (Table 7).
| EA | RP treatment during | No of oocytes examined | Autophagy activity (pixel/oocyte) in | ||
|---|---|---|---|---|---|
| Pa (4 hr) | IVC (24 hr) | One-cell oocytes before and after Pa | Two-cell oocytes after 24 hr of IVC | ||
| Before EAa | — | — | 53 | 1.00 ± 0.03a | — |
| After EAa | — | — | 55 | 1.10 ± 0.03b | — |
| Yes | No | — | 51 | 1.14 ± 0.03b | — |
| Yes | Yes (10 nm) | — | 53 | 1.24 ± 0.03c | — |
| Yes | Yes (100 nm) | — | 45 | 1.28 ± 0.02c | — |
| Yes | No | No | 43 | — | 1.00 ± 0.03 |
| Yes | No | Yes | 47 | — | 0.96 ± 0.01 |
| Yes | Yes | No | 42 | — | 0.98 ± 0.02 |
| Yes | Yes | Yes | 43 | — | 0.96 ± 0.02 |
- a–cWithin a column, values with different superscripts are different (p < .01).
- a Autophagy activity was examined before and immediately after EA, respectively.
4 DISCUSSION
Following fertilization, the maternal proteins of oocytes are degenerated, while the zygotic genome generates new proteins for embryonic growth (Menendez, Vellon, Oliveras-Ferraros, Cufi, & Vazquez-Martin, 2011). Although several proteins are degraded because of the ubiquitin proteasome system, autophagy has also been shown to play an important role in embryo metabolism during pre-implantation development (Menendez et al., 2011; Tsukamoto, Kuma, Murakami, et al., 2008). This process is important for cellular maintenance, cell viability and development, and is frequently related to type II programmed cell death in mammals (Baehrecke, 2003). The level of autophagy is lower in unfertilized oocytes than fertilized ones; however, autophagy is triggered immediately after fertilization, and this process plays an essential role in differentiation and development, as well as in the cellular response to stress (Shimada et al., 2011; Tsukamoto, Kuma, Murakami, et al., 2008).
It is difficult to produce pig embryos of homogeneous quality because of the relatively high incidence of polyspermy after IVF. Thus, diploid parthenotes have often been considered for analysis of early development in pigs (Paffoni, Brevini, Gandolfi, & Ragni, 2008; Tseng, Tang, & Ju, 2006). These reports lead us to study the effect of RP treatment during Pa and IVC on early development and to establish an IVC system using an autophagy inducer, RP, in pigs. Autophagy degrades the maternal mRNA and decreases apoptosis in pig parthenotes (Xu et al., 2012). In this study, we examined the effects of autophagy induction by treating oocytes with RP for various periods during Pa and/or IVC.
Among various concentrations of 1–100 nm RP, treatment of oocytes with 100 nm for 4 hr after EA augmented blastocyst formation of PA embryos compared to the control. Naturally, autophagy is high in IVM oocytes in pigs (Shimada et al., 2011; Xu et al., 2012) and activated immediately after fertilization. RP might synergistically increase the autophagy and worked beneficially for early embryo development, probably by stimulating embryo metabolism. In mice and pigs, autophagy is found to be high in activity from two- to four-cell embryos after PA (Tsukamoto, Kuma, Murakami, et al., 2008; Xu et al., 2012). Therefore, we treated PA embryos with various concentrations of RP during the first 24 hr of IVC to determine whether further stimulation of autophagy would influence embryonic development. RP at 1 and 10 nm significantly improved embryonic development to the blastocyst stage compared to control and 100 nm RP. These results indicated that higher doses of RP have no detrimental influence on early embryonic development, even after exposure for a relatively long time (24 hr). Conversely, 10 nm RP significantly increased embryo cleavage relative to the control and other concentrations of RP. Based on these results, we selected 10 nm RP for subsequent experiments. Our results were consistent with the previous findings obtained for mice and cattle in which 10 nm RP efficiently improved embryonic development (Shen et al., 2015; Song et al., 2012). Moreover, our study ensured the importance of autophagy induction during Pa and IVC for early embryonic development in pigs. The main role of autophagy in early embryonic development may be removal of maternally inherited proteins. Generally, maternal proteins are degraded after fertilization, and new proteins are generated from the zygotic genome (Fingar et al., 2004; Menendez et al., 2011; Xu et al., 2012).
We also evaluated the effects of RP during co-incubation of sperm and oocytes for 6 hr, the first 24 hr of IVC and both periods. Blastocyst formation of IVF embryos was increased by RP treatment during IVF, but not influenced by treatment during IVC. This finding was consistent with the results of a recent study in mice (Shen et al., 2015). Shen et al. (2015) also confirmed that RP accelerated active DNA methylation and removed the maternal mRNA. This activity of RP might improve the embryonic development. In contrast to PA embryos, the beneficial effects of RP on embryonic development after IVF were not observed when oocytes were treated with RP during IVC. However, RP treatment during IVF improved developmental competence of IVF embryos. Spermatozoal mitochondria and their DNA are not degraded by autophagy in IVF embryos (Luo et al., 2013). In addition, the activation process differs between PA and IVF embryos. Although the reasons for these different responses to RP treatment are not clear, this discrepancy might be due to different activation processes and reprogramming nature between them. Recently, the physiological role of autophagy and its underlying molecular mechanism have been evaluated intensely in zygote reprogramming. Maternal mRNAs and proteins are rapidly degenerated after the two-cell stage in mammalian embryos and new mRNAs and proteins are generated by the zygotic genome. This leads to changes in the proteins synthesized after the four- to eight-cell stages (Li, Lu, & Dean, 2013). Moreover, removal of maternal proteins and mRNAs would be necessary for activation of the zygotic genome (Alizadeh, Kageyama, & Aoki, 2005). The ubiquitin proteasome system plays an important role in removal of short-lived proteins during zygote remodelling, but long-lived proteins and organelles are degraded by autophagy (De Renzo & Seydoux, 2004; Tsukamoto, Kuma, & Mizushima, 2008). GSH is a ubiquitous cellular constituent that is the most available thiol-reducing agent in mammals. In a previous study, it has been reported that autophagy is enhanced by the overexpression of Atg5, an essential protein for autophagosome, and this Atg5 transgenic mice shows higher GSH concentration than wild-type mice (Pyo et al., 2013). The previous and our results indicate that GSH synthesis can be stimulated by enhanced autophagy. Furthermore, mouse embryonic fibroblasts were tolerant of protein oxidation and oxidative stress because of the enhanced autophagy induced by RP (Pyo et al., 2013). These beneficial actions of RP might have contributed to the improved embryonic developmental competence observed in our study.
An autophagy-related gene, Atg5, is important for early embryonic development because knockout of Atg5 suppresses embryonic growth at the four- and eight-cell stages. This occurs due to misleading in protein synthesis or recycling (Tsukamoto, Kuma, Murakami, et al., 2008). Thus, RP may help protein synthesis or recycling and remove the degraded or unwanted maternal materials. Unfertilized oocytes show a low level of autophagy than fertilized ones; however, autophagy is activated immediately after fertilization (Shen et al., 2015; Shimada et al., 2011; Song et al., 2012; Tsukamoto, Kuma, & Mizushima, 2008). A similar phenomenon was observed in this study that autophagy activity was increased in PA oocytes immediately after EA. This result indicates that the activation stimulus can be a trigger of autophagy induction even in PA oocytes. The autophagy level of activated oocytes remained in steady condition from 0 hr of EA up to 4 hr of Pa. RP treatment during Pa period additionally increased the autophagy level. Contrary to expectation, RP treatment during Pa and/or the first 24 hr of IVC did not alter the autophagy activity examined at 24 hr of IVC in PA embryos. Autophagy is transiently suppressed from the late one-cell to middle two-cell stages in fertilized mouse embryos (Tsukamoto, Kuma, & Mizushima, 2008). In pigs, microtubule-associated protein 1-light chain 3 mRNA transcription drops rapidly after one-cell stage in PA oocytes (Xu et al., 2012). Based on these previous findings, it was considered that autophagy induction by RP treatment during Pa and/or IVC might be masked by the physiological suppression of autophagy around two-cell stage embryos. It remains unknown in this study how or why embryonic development is improved after RP treatment during IVC despite no increase in autophagy activity. The GSH content was increased by the RP treatment during Pa and remained high even after 24 hr of IVC. It was probable that the high level of GSH content contributed to the improved blastocyst formation.
In summary, it seemed in this study that early induction of autophagy by RP treatment during Pa or IVF might have improved embryonic development. Our results demonstrate that RP plays a positive role on early embryonic development in pigs by increasing autophagy activity although the positive effect is shown differently between IVF and PA embryos probably due to different activation processes between them. Further study is needed to clearly elucidate the possible mechanisms of autophagy induction after RP treatment during Pa and early stage of embryonic development.
ACKNOWLEDGEMENTS
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (Grant No. 2015R1A2A2A01005490) and the Bio-industry Technology Development Program (IPET 31060-05-1-CG000), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
CONFLICT OF INTEREST
None of the authors have any conflict of interest to declare.
AUTHOR CONTRIBUTIONS
FE, SH and EL designed the study, while FE, HS and HL performed the experiments. FE and JL assayed autophagy activity. The manuscript was written and corrected by FE, SL, CP and EL.
