Effects of pregnancy serum and scriptaid on development in early partheno embryo

Partheno Embryo's research is known to play a very important role in identifying the development of embryonic cells or analyzing the genetic mechanisms of embryonic development, but the information on apoptosis formed during the early stage of development on Partheno Embryo is very little. Therefore, this study analyzed whether the embryonic cell death of unit embryos can be inhibited by adding Scriptaid, one of HDACi, which plays a role in demethylation of histone proteins as a method of regulating the cell cycle in the early embryo development of Partheno Embryo. As a result, the differentiation rate was higher in the group that added Scriptaid and FBS, but the cellular development was higher in the group that added pregnant serum to Scriptaid. As a result of analyzing the expression of the gene through IF and PCR, the group with the addition of gestational serum increased the expression of BCL2 and PCNA, which affects the anti-Casp3 action in cell survival. In addition, it is interpreted that treatment of Scriptaid for 16 hours, rather than 24 h treatment lowers the expression of Casp-3, a representative factor of apoptosis, and also increases embryonic development, thus affecting early embryo development. Therefore, it is concluded that the 16-hour treatment of Scriptaid and the use of gestational serum will inhibit cell death in the early embryonic development and increase the development rate of the embryo.

INTRODUCTION through the gene activation of PKA and PKC through the expansion of Ca + Channel into the cytoplasm, and this phenomenon is mistaken for the normal nuclear fusion process of sperm and starts cell division (Russo et al., 1996). However, due to the lack of the male gene, the early development of partheno embryos is inhibited in metabolism compared to normal in vitro fertilization, and anomalies due to starvation and external shock of cells are formed, which develop into apoptosis (Cha et al., 1997). The apoptosis mechanism can largely explain two mechanisms of apoptosis and autophagy. Among them, the apoptotic phenomenon, which is an internal necrosis action, is representative, and it can be regarded as a basic apoptosis process due to the starvation state and external impact of the cells (Lee et al., 2011). Particularly, due to the nature of partheno embryo, physical stimulation is known to induce cell death by controlling the metabolic action of mitochondria in the cell cycle. And the action of IGF2 is inhibited in the HDACi conjugate composition, causing starvation of the cell (Elahi et al., 2018;Lee et al., 2018).
In addition, it is thought that the activity of Caspase-3 plays a direct role in apoptosis due to the formation of an apoptotic complex (Ryu and Yoon, 2018). In other words, the inhibition of the development of partheno embryos can be judged to be accelerated by apoptosis, as the occurrence of metabolic stimulating factors that are formed in the early or middle of cell division is inhibited by external apoptosis stimulating factors. According to a recent study as a control method for such a phenomenon, the use of scriptaid is suggested as a way to increase the metabolic process by facilitating the generation of embryonic cells in the cell division process (Diao et al., 2013;Jungmin et al., 2013). Scriptaid is a HDACi (Histone Deacetylase inhibitor) that affects various processes such as growth retardation, differentiation, cytotoxicity, and apoptosis, and modifies the expression of abnormally expressed genes (Yang et al., 2014). It is also known to increase the action of IGF signal necessary for cell growth by acetylating Histone protein, which maximizes embryo development (Duncan et al., 2017). This study is to determine whether the action of scriptaid may inhibit the cell death process in the early development of unit embryos, and in the in vitro culture process, using normal serum and serum of the early gestation period, maximizing the mechanism of metabolism.

In vitro maturation
Porcine ovaries were collected at local abattoirs (DODRAM, Ansoung, Korea) and transported to the laboratory in 0.9% physiological saline (with Penicillin G 100 IU/mL, Streptomycin 100 μg/mL) at 37 o C within 2 hr of slaughter. Cumulus-oocyte complexes (COC) were aspirated from surface ovarian follicles 3-6 mm in diameter using an 18-gauge needle attached to a 10-mL disposable syringe. The supernatant was removed and the precipitate washed three times in a wash medium containing Hepesbuffered tissue culture medium 199 (TCM-199), 10 μL/mL antibiotic and 0.3% (w/v) bovine serum albumin (BSA).

Parthenogenetic activation
After a 44 hours IVM period, oocytes were released from follicle cells by treatment with 0.1% hyaluronidase. The removed oocytes were washed several times in an activation solution (0.28 M D-mannitol, 0.1 mM MgSO 4 , 0.05 mM CaCl 2 and 0.01% BSA) and then activated with a direct current chemical stimulus at 7 min using 7% ethanol.
After oocytes activation was induced for 2 hours in 2 mM 6-DMAP (6-dimethylaminopurine), and then stabilized by incubating for 4 hours in 2 Mm H-DSOF (HEPES-buffered synthetic oviduct fluid) to promote cell development.

In vitro culture
For experiments using in-vitro culture, we prepared three types of in-vitro culture media: FBS group medium is the NCSU-23 medium (North Carolina State University 23 medium) contained 10% FBS (Petters and Wells, 1993), PS group medium is the NCSU-23 medium contained 10% Pregnancy serum (Cow blood serum after 30 days of pregnancy), scriptaid was added to 1 mM and incubated for 16 hours or 24 hours, respectively, and then replaced with a scriptaid free medium. Ten zygotes were added to 30 μL drop of each type of prepared medium and covered with mineral oil, incubated at 39 o C for 4 days in 5% CO 2 , humid atmosphere. The zygotes were incubated for 3 days, then transferred to freshly prepared culture medium. Subsequently, cell division rates were evaluated on culture days 2, 3 and 4, respectively (Visual analysis was performed according to the methods of Alikani et al., 1999 andMateusen et al., 2005).

Enzyme linked immunosorbent assay
For ELISA, total protein was extracted from parthenogenesis embryo using Pro-Prep solution (Intron) according to the manufacturer's instructions. Total protein was quantified using the Bradford Protein Assay Kit (Bio-Rad).
For quantification of specific proteins from culture medium and cellular proteins, samples diluted in assay buffer were used to coat a 96-well ELISA plate overnight at

Zymography
In vitro culture medium is added to Foz loading buffer (0.06% bromophenol, 10% SDS, 2% glycerol), left on ice for 5 minutes, and then electrophoresed at 150 V for 1 hour 30 minutes; A 12% SDS-PAGE gel containing 100 mg/mL gelatin A/B was used. The gel was washed twice with reconstruction buffer (2.5% Triton X-100) for 20 minutes, then placed in a zymography reaction buffer and incubated at 37 o C for 18 hours. Then, the gels were stained (0.5% Coomassie Blue R250 (Bio-rad, USA)) for measurement of white band to MMP-2 and MMP-9 activity. Average fold values for all hormones were measured and are expressed as mean ± standard deviation. Data were analyzed using t-test and generalized linear model method using SPSS Statistics 20 (SPSS, Korea).

Statistical analyses
The real-time RT-PCR results were analyzed for statistical significance using the SAS package (version 9.4; Statistical Analysis System Institute, Cary, NC, USA). Data were subjected to Welch's t-test, fold change, and GLM in SAS. Data are the mean ± SD, and differences between groups were considered significant when p values were less than 0.05.

Changes in embryonic cells during in vitro culture
The FBS group showed higher development rate than the pregnancy serum group, and the Scriptaid treated for 16 hr group had a higher development rate than the Scriptaid treated for 24 hr group (Table 2).
In addition, in the treatment of Scriptaid was higher cytoplasm degradation in the 24 hr treatment group compared to the 16 hr treatment group.
On the other hand, when Scriptaid was treated with pregnancy serum, it was confirmed that the increase in the degradation were suppressed than when using FBS.
In particular, the group treated with Scriptaid 16 hr and pregnancy serum showed a decrease in degradation compared to the group treated only with pregnancy serum (Fig.   1).

MMPs expression in embryonic cells
When the scriptaid treatment time was 24 hr, the expression of MT1-MMP increased slightly, while in the case of MMP-2, it tended to decrease, but it did not show a significant difference and showed similar overall expression level. The activity of MMPs was different depending on the exposure time of the scriptaid and the type of serum. In particular, in the group using FBS with scriptaid treatment, the activity of active MMP-9 was low, and the activity of MMP-2 was high.
On the other hand, in the group supplemented with scriptaid and pregnancy serum, the activity of MMP-9 dimer was shown, and the activity of MMP-2 was slightly lower than that of the FBS added group (Fig. 2).

Analysis of gene expression patterns related to cell survival and apoptosis
In the case of PCNA and E2-r, factors related to cell survival, high expression was observed in the pregnancy serum group treated with 16 hr scriptaid. In addition, the anti-apoptosis factor BCL-2 was the highest in the 16 hr scriptaid treated pregnancy serum group, and the expression of BAX was lower than from the other groups (Fig. 3). As a result of analyzing the expression of Casp-3 and PCNA through IF, the expression of Casp-3 in the cytoplasm was high in the group treated with scriptaid.  and it is known to be focusing on stem cell production by autogenous replication recently (Brevini and Gandolfi, 2008). However, it is a common phenomenon that a partheno embryo does not induce implantation due to the absence of specific genetic action of the paternal line (Surani et al., 1984). Particularly, in the early embryonic development, the development of the partheno embryo is lower than that of the normal fertilized embryo, thereby increasing the mechanism of cell death due to starvation (Cha, 1997). In this study, we observed the abnormality of cells in the early embryonic development period of the partheno embryo, also, analyzed whether serum changes and Scriptaid, which plays a role in the demethylation of histone proteins in the cell cycle, can control the starvation and apoptosis. As a result of analyzing the normality of partheno embryo cells, this study confirmed that the difference in serum affects the differentiation process of partheno embryo differently. In the case of partheno embryos induced differentiation using FBS, the development rate of blastomere was relatively low, and particularly, it was confirmed that degradation were highly formed between the cytoplasm and the cell membrane. However, in the case of partheno embryos cultured using pregnency serum, it was confirmed that differentiation and fidelity of the cytoplasm increased (Jung et al., 2020).
The results of this study suggest that the addition of  (Park et al., 2017) can be reduced by the use of pregnency serum and scriptaid. In particular,

CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was reported.