Effects of Cell Status of Bovine Oviduct Epithelial Cell ( BOEC ) on the Development of Bovine IVM / IVF Embryos and Gene Expression in the BOEC Used or Not Used for the Embryo Culture

The objective of this study was to investigate the effects of cell status of BOEC on development of bovine IVM/IVF embryos and gene expression in BOEC before or after culturing of embryos. The developmental rates beyond morula stage in the BOEC co-culture group was significantly higher than in the control group (p<0.05). In particular, blastocyst production in the BOEC co-culture group (28.3%) was dramatically increased compared with the control group (7.2%). In the in vitro development of bovine IVM/IVF embryos according to cell status, the developmental rates beyond morula stage in the primary culture cell (PCC) co-culture group were the highest of all experimental groups. Expression of genes related to growth (TGF-β, EGF and IGFBP), apoptosis (Bax, Caspase-3 and p53) and antioxidation (CuZnSOD, MnSOD, Catalase and GPx) in different status cells of BOEC for embryo culture was detected by RT-PCR. While EGF gene was detected in isolated fresh cells (IFC) and PCC, TGF-β and IGFBP were found in IFC or PCC after use in the embryo culture, respectively. Caspase-3 and Bax genes were detected in all experimental groups regardless of whether the BOEC was used or not used in the embryo culture. However, p53 gene was found in IFC of both conditions for embryo culture and in frozen/thawed culture cells (FPCC) after use in the embryo culture. Although antioxidant genes examined were detected in all experimental groups before using for the embryo culture, these genes were not detected after use. This study indicated that the BOEC co-culture system used for in vitro culture of bovine IVF embryos can increase the developmental rates, and cell generations and status of BOEC might affect the in vitro development of bovine embryos. The BOEC monolayer used in the embryo culture did not express the growth factors (TGF-β and EGF) and enzymatic antioxidant genes, thereby improving embryo development in vitro. (


INTRODUCTION
Since the birth of the first calf derived from in vitro fertilization (Brackett et al., 1982), considerable progress has been made in the development of techniques for in vitro production (IVP) of bovine embryos for both research and commercial purposes.In particular, in vitro culture systems using somatic cells were paid attention by many investigators for the IVP system in bovine embryos.The techniques for IVP of bovine embryos for breeding or research purposes require that the culture system supports high embryo development with repeatable results (Kim et al., 2007).However, developmental rates to blastocysts for in vitro culture systems remain low and range between 20-30 percent (Carolan et al., 1996;Kreysing et al., 1997).
Several types of somatic cell have been used for in vitro culture of bovine embryos including bovine oviduct epithelial cell (BOEC), granulosa cell, uterine fibroblasts, trophoblastic vesicles and buffalo rat liver cells (Orsi and Reischl, 2006).The co-culture system with somatic cells is an effective and useful method for in vitro culture of bovine IVF embryos, affecting the success of IVP systems which may stimulate embryo development through amelioration of embryotoxic substance or secretion of embryotropic factor (Carolan et al., 1995;Donnay et al., 1997).Antioxidant enzymes (MnSOD, CuZnSOD, Gpx, glutamylcysteine synthetase (GCS) and catalase) as an embryotropic factor were constituently expressed in both bovine oviduct cell monolayers and nonattached epithelial cell vesicle cultures during an 8-day culture period (Harvey et al., 1995;Jang et al., 2005).The somatic cells used to increase development in the embryo co-culture system may secrete various growth factors, including IGF, LIF and TGF (Rief et al., 2002).BOEC are the most popular and notable somatic cells in bovine embryo culture in vitro.The BOEC co-culture system is an excellent method for overcoming the in vitro developmental block of bovine IVM/IVF embryos.However, BOEC may produce unknown substances, embryotropic factors and/or embryotoxic factors, making in difficult to determine the exact requirements for embryo development and preventing a clear understanding of embryo metabolism.Moreover, the effects of status (more than 1-passage and fresh or frozen/thawed) of BOEC on in vitro development of embryo culture has not been investigated and also the specific mechanism of their action is still unclear.Therefore, more complete analysis of the somatic cell co-culture system supporting high developmental rates is needed, not only to obtain consistent results but also to examine the exact requirements of embryo culture in vitro.Furthermore, cell lines that retain the ability over many passages to provide the conditions favorable for embryo development through the transition stage should be established.
The goal of the present study was to investigate the factors affecting increased in vitro developmental rates when Hanwoo IVM/IVF embryos were co-cultured with BOEC.The present study was designed to determine 1) the effects of the BOEC co-culture system on in vitro development rate of bovine IVM/IVF embryos, 2) the effect of status of BOEC on in vitro development of bovine embryos, and 3) detection of the temporal patterns of expression of growth factor genes (TGF, EGF and IGFBP), antioxidant genes (Cu/Zn-SOD, Mn-SOD, Catalase and GPx) and apoptosis genes (Bax, p53 and Caspase-3) in different types of BOEC co-cultured with or without bovine IVM/IVF embryos.

In vitro production of bovine IVM/IVF embryos
The cumulus-oocyte complexes (COCs) aspirated from the ovaries obtained from an abattoir were matured in TCM 199 (Gibco, USA) that was supplemented with 10% fetal bovine serum (FBS, Gibco), 1 μg/ml estradiol-17β, 5 μg/ml luteinizing hormone and 0.5 μg/ml follicle stimulating hormone at 38.5°C in humidified 5% CO 2 in air.Following maturation, 10-15 matured oocytes were transferred into a 50 μl fertilization drop (BO medium; Brackett and Oliphant, 1975).Frozen/thawed Hanwoo semen was washed in BO medium containing 10 mM caffeine and 50 μl sperm suspension was introduced into each fertilization drop.Presumptive zygotes produced at 6-8 h after insemination were cultured for 34-36 h in CR 1 aa medium and 2-and 8cell embryos were randomly allocated into each experimental group.Embryos were cultured for 5-6 days in 5% CO 2 in humidified air at 38.5°C and were examined for appearance, stage of development and cell number in blastocysts following Hoechst 33342 staining.All chemicals used in this study were purchased from Sigma-Aldrich (USA) unless otherwise stated.

Isolation, primary and secondary culture of bovine oviduct epithelial cell (BOEC)
Bovine oviducts were transported on ice from the slaughterhouse to the laboratory within 2 h.The oviducts were carefully trimmed on ice and rinsed with phosphatebuffered saline containing 1% antibiotic and antimycotic solution (Gibco).The epithelial cells were dispersed by forcing medium through an 18-gauge needle attached to a 10ml syringe and then the samples were transferred into 50 ml conical tubes containing 25 ml of Dulbecco's modified eagle medium (DMEM, Gibco) containing 10% FBS for washing twice by centrifugation (200×g) at 4°C.Cells were adjusted to 3×10 6 cell/ml with DMEM containing 10% FBS.The cell pellet was then equally dispensed as 1 ml volumes into a 4-well tissue culture dish and cultured at 38.5°C.In 5% CO 2 in humidified air, and the fresh medium changed every two days.Formation of a confluent monolayer with some ciliary activity within 2-3 days was affirmed and cultured with or without embryos for 6-7 days, and the other one was used for secondary-culture or cryopreservation.

Freezing and thawing methods of BOEC
BOEC monolayer was dissolved with Trypsin-EDTA (Gibco).After washing of the cells by centrifugation (500×g, 10 min), the supernatants were removed and cell pellets were frozen for cryopreservation.Basic freezing medium was used with DMEM containing 20% FBS.Dimethyl sulfoxide (DMSO) was then slowly added to reach a final concentration of 10% of the total volume.The cell suspension was dispensed into freezing vials and then placed in a -70°C freezer overnight.Freezing vials were plunged directly into liquid nitrogen on the next day and stored until use.Frozen BOEC was thawed at 37°C for 30 sec and used either for embryo culture or gene detection.

Morphological analysis
Following the different cell status, the morphology of BOEC was evaluated by visual examination under phasecontrast microscopy.Another morphological examination using a fluorescence staining procedure with 4', 6'diamidino-2'-phenylindole (DAPI) was performed as described previously (Yim et al., 2000).Briefly, the BOEC was cultured in 1-chamber slides at a density of 2×10 4 cell per chamber.The cells were washed twice with PBS and fixed by incubation in 4% paraformaldehyde for 30 min.The fixed cells were again rinsed twice with PBS and then incubated in 1 μg/ml DAPI solution for 30 min.The apoptotic cells were observed under epifluorescence microscopy equipped with an excitation/barrier filter of 280-365 nm and a digital camera.

RNA isolation of BOEC
Total RNA was also extracted by the guanidium isothiocyanate method (Chomczynski and Sacchi, 1987) from different status of BOEC or from BOEC monolayer which had been used or not used for embryo culture.Oviduct cells were placed into extraction solution (0.025 M sodium citrate, 0.5% sarcosyl, 4 M guanidiumthiocyanate, pH 7.0) and were vortexed with phenol, chloroform:isoamyl alcohol (24:1) and 2M NaOAC, centrifuged at 20,000×g at 4°C for 20 min and the supernatant transferred to a new tube.After addition of isopropanol and gently shaking, the supernatant was then precipitated with isopropanol at -20°C overnight.The samples were then centrifuged, washed with 70% ethanol, recentrifuged and air-dried before being dissolved in diethylprocarbonate (DEPC)-treated distilled water.The yield of extracted total RNA for each sample was determined by UV spectrophotometer at 260 nm.

Reverse-transcription polymerase chain reaction (RT-PCR) analysis
Each mRNA collected in the total RNA sample was analyzed by RT-PCR.Reverse-transcriptase generation of cDNA was performed on 2 μg of total RNA in a final volume of 2 μl with reverse transcription reagents (RT mixture), RT buffer, 0.5 mM of each dNTP, 0.5 μg oligo (dT), 10 IU RNase-inhibitor (Gibco) and 500IU reverse transcriptase (Ambion).The reaction was carried out at 42°C.for 60min followed by denaturation for 10 min at 98°C and the remaining RT products were stored at 4°C.Subsequent PCR analysis was performed on 2 μl of the cDNA in a final volume of 20 μl; the PCR mixtures consisted of PCR buffer, 0.2 mM dNTP, 10 μM primer and 2 IU Taq polymerase.PCR was carried out for 3 min at 95°C, 50 sec at 60°C, 1 min at 72°C and 30 cycles.Primer sequences used in this study are indicated in Table 1.For each sample, PCR amplification products were analyzed on 2.0% agarose gel stained with 0.5 μg/ml ethidium bromide, visualized under UV light and photographed.Standard DNA markers (1 kb DNA ladder) were also used to determine the size of amplified products.

Statistical analysis
The SAS mixed linear model program was used to analyze the data.Percentage of developmental stages in each treatment was compared for differences through use of Duncan's modified multiple range tests.Values at 5% level or less (p<0.05) were considered statistically significant.

Effects of BOEC co-culture system on bovine IVM/IVF embryos
The effects of different status of the BOEC co-culture There was no difference in mean cell number of blastocysts between groups (Table 2).In the bovine IVM/IVF embryos cultured with different BOE cell generations and status, the developmental rates to blastocysts in PCC (40.6%±5.0)and secondary culture cell (SCC, 30.8%±6.2) were higher than in the control (20.9%±2.9)and FPCC groups (14.7%±4.6).
The FPCC group gave rise to fewer blastocysts among the experimental groups, but mean cell number of blastocysts was not different among the experimental groups.

Morphology and gene expression in different status of BOEC used or not used in embryo culture
The influence of different cell status on cell morphology was evaluated from visual examination by phase-contrast microscopy and DAPI staining under eqifluorescence microscopy.In Figure 1, FPCC showed retraction of membranes, cytoplasmic condensation and irregularity in shape when compare to PCC and some nuclei of the FPCC exhibited typical features of apoptosis, such as nuclear condensation and nuclear fragmentation, however, the SCC lessened these typical features of apoptosis.
To establish an mRNA phenotypic map for the expression of various growth factors, apoptosis and antioxidant genes, BOEC used or not used in embryo culture were analyzed according to different cell status by RT-PCR.The expression patterns for growth factor genes in BOEC with different status which was used or not used in embryo culture are shown in Figure 2. TGF-β and EGF genes were expressed in IFC, whereas another EGF gene was only expressed in PCC.Transcription for IGFBP was only expressed in PCC after use in the embryo culture.
The expression patterns of apoptosis genes (Bax, Caspase-3, and p53) in BOEC with different cell status, which was used or not used in embryo culture, were examined (Figure 3).Expression of p53 was detected in IFC regardless of embryo culture and was only detected in FPCC after use in the embryo culture.On the other hand, Bax and Caspase-3 genes were expressed in all treatment groups regardless of embryo culture.
The transcripts for CuZnSOD, MnSOD, Catalase, and GPx were detected in BOEC used or not used in embryo culture according to different cell generation and status   (Figure 4).All antioxidant genes were observed in BOEC groups that were not used in embryo culture.In contrast, the antioxidant genes were not expressed in BOEC groups after use in the embryo culture.

DISCUSSION
Mammalian preimplantation embryos produced or cultured in vitro commonly exhibit both short-and longterm retarded development (Kim et al., 2004), reduced viability (Gardner and Lane, 2005) and developmental arrest (Jurisicova and Acton, 2004).Mammalian preimplantation embryos for in vitro culture inevitably face the deleterious conditions that retard or arrest the development of embryos in vitro.The deleterious conditions for in vitro culture of implantation embryos are the exposure of ROS by high oxygen tension, heavy metal ions, unknown embryotoxic substances and others.
Recent attention has focused on ROS as major factors of in vitro embryonic arrest and cell death on in vitro development (de Lamirane and Gagnon, 1992;Guerin et al., 2001).The generation of ROS is an essential prerequisite for the normal function of many cells, however, excessive formation can lead to cellular damage and pathology (Halliwell and Aruoma, 1991).ROS exert a powerful oxidizing potential and have a pronounced effect on DNA, RNA and protein synthetic activities (Nasr-Esfahani et al., 1992;Johnson and Nasr-Esfahani, 1994).The deleterious effects of ROS are counter-balanced by antioxidant that neutralizes the activated oxygen species.
To overcome the in vitro block events or deleterious action of ROS, preimplantation embryos produced either in vitro or in vivo were cultured in a somatic cell co-culture system, with supplementation of specific compounds such as thiol compound, macro-molecules, growth factors and antioxidants.The somatic cell co-culture system has been used by many researchers to enhance in vitro development, to overcome cell block events and to delay apoptosis (Harvey et al., 1995;Vanroose et al., 2001).The specific antioxidant enzymes are produced by cells and are able to detoxify O 2 , H 2 O 2 , and organic peroxides.These enzymes include MnSOD and CuZnSOD, which catalyze the dismutation reaction, removing O 2 species; catalase, which catalyses the decompositon of hydrogen peroxide to oxygen and water; and Gpx, which utlilzes glutathione as a reducing source to catalyze the removal of both hydrogen  peroxide and lipid peroxides (Johnson and Nasr-Esjahani, 1994).However, the exact mechanism of somatic cell coculture system on development of preimplantation embryos in vitro is still unclear.
This study examined the effects of BOEC co-culture system on the development of bovine IVM/IVF embryos.We found that BOEC co-culture system improved significantly the development rates beyond morula stages when embryos were cultured in PCC.Bavister (1992) reported that the somatic cell co-culture system could increase the developmental rate of embryos in vitro, including overcoming the developmental block, increasing the number of embryos reaching the blastocyst stage and improving the viability of embryos following transfer.Our results were consistent with the previous findings that coculture with somatic cells could enhance in vitro development of embryos (Bavister, 1992;Harvey et al., 1995;Liu et al., 1998).In addition, we examined whether the different cell status of BOEC co-culture system affected in vitro development of bovine IVM/IVF embryos.Studies evaluating the use of fresh or sub-passaged monolayer indicated that previously sub-passaged cell monolayer might provide a more uniform and consistent co-culture system for mammalian embryos (Ouhibi et al., 1990).Furthermore, other researchers have evaluated in vitro development of ovine embryo after using either primary or frozen/thawed oviduct cells for co-culture (Rexroad and Powell, 1988).They reported that there was no significant difference between the two sources of oviduct cells in their ability to support in vitro development of one-cell ovine eggs.However, developmental rates from our results were significantly higher when embryos were cultured in PCC than those of control, SCC and FPCC groups (p<0.05).Our results agreed with the results of Ouhibi et al. (1990), but disagreed with those of Rexroad and Powell (1988) considering the difference in embryo source and methodology used by different laboratories.Our results indicated that the status of BOEC largely affected the development of bovine IVM/IVF embryos.It is suggested that fresh oviduct cell monolayers in the embryo co-culture system may indirectly provide evidence indicating that the embryotropic factors secreted by somatic cells can enhance embryo development in vitro.
The somatic cells used to increase the in vitro development of preimplantation embryos during in vitro culture may secrete embryotropic factors, which may include specific proteins such as glycoprotein 85-97 and various growth factors, including IGF, LIF, TGF, and some kinds of antioxidants (Rief et al., 2002).The antioxidant enzymes that regulate the ROS generated during in vitro culture of preimplantation embryos play a critical role in protection of somatic cells from oxidative stress-induced cell death.However, when embryo and somatic cells during in vitro culture overcome the block system and the antiapoptotic system, cells begin programmed cell death, namely apoptosis.Genes of the caspase family, p53, Bax and cytochrome c, are relative to apoptosis in cell life.Nuclear translocation of p53 and transcriptional activation of Bax and Fas (Muller et al., 1998;Lee et al., 2007) are known to be induced by H 2 O 2 (Uberti et al., 1999) and p53induced apoptosis requires the generation of ROS (Li et al., 1999).We detected different patterns of mRNA expression for growth factors, antioxidants and apoptosis genes on different status of BOEC used or not used in embryo culture.Expression of growth factor genes and TGF-β gene was detected in IFC and EGF gene was detected in IFC and PCC before use in embryo culture.Furthermore, transcripts for IGFBP were only expressed in PCC cultured with bovine IVM/IVF embryos.Apoptosis genes such as Bax and Caspase-3 were observed in all experimental groups irrespective of the embryo culture.On the other hand, transcription for p53 was only detected in IFC used or not used in embryo culture and FPCC after use in the embryo culture.Expression of antioxidant genes was observed in all groups of BOEC before use in embryo culture, while this expression was not detected in BOEC after use in embryo culture.This study indicated that growth factors and antioxidants might influence the development of bovine embryos in vitro, indicating that both genes were only detected in fresh cells or fresh and frozen/thawed BOEC monolayer before use in embryo culture.
Therefore, our results demonstrated that the co-culture system of bovine oviduct epithelial cell according to cell status could increase the developmental rates of bovine IVM/IVF embryos through its antioxidant activity and the secretion of embryotropic factors.

Figure 2 .
Figure 2. mRNA expression of growth factor genes in different cell status of BOEC used or not used in embryo culture (1, Isolated fresh cell; 2, Primary culture cell; 3, Secondary-culture cell; 4, Frozen/thawed primary culture cell; 5, Primary culture cell after use in embryo culture).

Table 1 .
Oligonucleotide primer sequence for growth factors, antioxidant and apoptosis genes system on development of bovine IVM/IVF embryos were examined.The difference of developmental rates in Table2and 3 was considered from results of independent experiments.The developmental rate of blastocysts in the BOEC co-culture group (28.3%±0.7)was almost three times than that of the control group (7.2%±1.3,p<0.05).

Table 2 .
Effect of BOEC co-culture system on the development of bovine IVM/IVF embryos

Table 3 .
Effects of the generation and status of BOEC on development of bovine IVM/IVF embryos