Effects of IGF-I and EGF Supplemented to PZM 3 Culture Medium on the Development of Porcine Embryos In vitro

This study investigated the effects of IGF-I and EGF on the development of blastocysts or hatched blastocysts during the in vitro culture of embryos from immature porcine oocytes. After the in vitro maturation and fertilization of cumulus-oocyte complexes (COCs) and their culture in vitro in PZM3 medium, we examined the embryo development rate for 168 h. When different concentrations of IGF-I (0, 1, 10, 20 ng/ml) were supplemented to fertilized porcine embryos in vitro, there were no significant differences in cleavage rate, blastocyst development rate or blastocyst hatching rate among the treated groups. On the other hand, when different concentrations of EGF (0, 1, 10, 20 ng/ml) were supplemented to the in vitro culture medium, blastocyst development rate was highest in the group in which EGF was not supplemented and, specifically, it was higher than in the 20 ng/ml treatment group (p<0.05). When 10 ng/ml IGF-I and 1 ng/ml EGF were supplemented separately or simultaneously, there were no significant differences among the treated groups in blastocyst hatching rate and the number of cells in each condition. This study demonstrated that the addition of IGF-I and EGF into PZM3 medium did not enhance development of the blastocyst stage and total cell number in blastocysts. (


INTRODUCTION
The in vitro production of porcine embryos has been an important basis for the study of the generation process of mammalian embryos; furthermore, it is widely adopted for cutting-edge biotechnologies such as transgenetic animal cells, development of micromanipulation (Lee et al., 2007), and stem cell studies, etc.
In general, the in vitro production of mammalian embryos is generally classified into maturation, fertilization and culture of embryos.Also, the system of in vitro embryo production technologies has been founded on the production of a number of offspring through the transplantation of in vitro-produced blastocysts (Kikuchi et al., 2002).However, in the case of the porcine species in particular, as compared with other animal species, nuclear maturation and cytoplasmic maturation are not coordinated, so it is difficult to obtain good quality embryos due to unstable in vitro maturation (Wang et al., 1997), unstable male pronucleus production (Motlik et al., 1984), polyspermy during in vitro fertilization (Abeydeera and Day, 1997), and developmental delays or stoppage phenomena while in the four-cell stage during in vitro culture (Camous et al., 1984;Heyman et al., 1987;Uhm et al., 2009).In order to overcome these problems, numerous studies have been conducted regarding the relevant factors for in vitro culture using immature porcine oocysts, specifically on topics such as the optimization of culture solution components (Kim et al., 1993), the presence of co-culture with somatic cells (Xu et al., 1992), static/perifusion culture systems (Lim et al., 1996;Kim et al., 2007), supplementation of growth factor (May, 1988;Chang et al., 2002;Makarevich et al., 2006) and supplementation of antioxidant (Ali et al., 2003;Lee et al., 2004).
In general, in the culture of porcine embryos Whitten's medium, Beltsvile embryo culture medium3 (BECM-3), North Carolina State University23 (NCSU-23) and Porcine zygote Medium 3 (PZM3) are used.Among these, PZM3 is most commonly used and has recently resulted in better outcomes than any other medium (Yoshioka et al., 2002).The culture medium for embryos is composed of simple minerals, energy sources, amino acids, pH buffers, trace elements and antibiotics.Recently, there have been continuous studies on the effects on embryo development of growth factor, antioxidants and chelators supplemented to basic culture medium.
It was reported that the growth factor, one of the most important materials that are supplemented to an in vitro culture medium, controls proliferation, differentiation and shape of mammalian embryonic cells in vivo (Hill et al., 1992).The growth factor also has the ability to promote the development of endometrium and embryo by controlling the functions of the ovary and uterus during the gestational period (Adashi et al., 1985;Echternkamp et al., 1994).In addition, it promotes embryo development in mice (Demeestere et al., 2004), rabbits (Herrler et al., 1998), cattle (Narula et al., 1996;Moreira et al., 2002), and humans (Lighten et al., 1997), as well as increasing the overall cell numbers of blastocysts produced.Among the growth factors, insulin-like growth factor I (IGF-I) maintains the pregnancy with the interaction between endometrium and embryo (Simmen et al., 1993), and it plays an important role in inhibiting the cell death of various cell types such as hematopoietic cells (Minshall et al., 1996), fibroblasts (Kulik et al., 1997) and oophytes (Morita et al., 1999).
In contrast, regarding the cytoplasmic maturation of immature mouse, cow and human oocytes (Harper and Brackett, 1993;Park and Lin, 1993), EGF is effective for nuclear membrane disruption (Down et al., 1989;Das et al., 1991) and expansion of cumulus cells (Larson et al., 1990).Furthermore, it was reported that in mice, during the in vitro culture of embryos, EGF promoted the blastocoele formation (Wood and Kaye, 1989), and it also increased the blastocyst development rate of bovine and porcine embryos (Harper and Brackett, 1993;Wei et al., 2001).Although there have been comparisons between the effects of IGF-I and EGF on embryo development as well as hatching of porcine embryos, there have been no reports as yet regarding the effects of PZM3.
Therefore, in this study, the effects of IGF-I and EGF supplemented to PZM3 culture medium on the development of in vitro porcine embryos were examined in order to improve the understanding of culture conditions for the basal molecular metabolic processes involved in the in vitro production of porcine embryos.

Chemicals and media
Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chemical Co.(St, Louis, MO, USA).Solutions are expressed as percent dilutions (v:v) and all media used for IVM, IVF, and in vitro culture (IVC) were pre-warmed to 39°C in a 5% CO 2 incubator with maximum humidity for 4 h before use.

Oocyte collection and in vitro maturation
Ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory in saline supplemented with 25 μg/ml gentamicin at 25-30°C within 2-3 h.Cumulus oocyte complexes (COCs) were obtained by aspiration from follicles 2 to 6 mm in diameter using an 18gauge needle connected to a 10-ml disposable syringe.Only COCs with compact cumulus cell layers and evenly granulated ooplasm were selected.The COCs were washed three times in HEPES-Tyrode-albumin-lactate-pyruvate medium (TALP medium) supplemented with 25 mM HEPES and 3 mg/ml BSA.Groups of 50 COCs were placed into 500 μl of BSA-free NCSU-23 solution with 0.57 mM cysteine, 10% porcine follicular fluid (pFF), 2.5 mM βmercaptoethanol, 10 ng/ml epidermal growth factor (EGF), 10 IU/ml human chorionic gonadotropin (hCG) and 10 IU/ml pregnant mare serum gonadotropin (PMSG) in each well of a 4-well multidish (Nunc, Roskilde, Denmark).After 22 h for maturation, oocytes were washed twice in the same maturation medium without PMSG and hCG and cultured in this medium for 22 h at 39°C in an atmosphere of 5% CO 2 and maximum humidity.

Sperm preparation and in vitro fertilization
Diluted porcine semen was produced by Dabby A.I. Center and stored at 17°C for 5 days.The semen was layered on top of a discontinuous Percoll density gradient (2 ml of 45% Percoll over 2 ml of 90% Percoll) in a 15-ml centrifuge tube.The sample was centrifuged for 20 min at 500×g at room temperature.The spermatozoa collected in the bottom fraction were washed three times: twice in D-PBS containing 1 mg/ml BSA, 100 μg/ml penicillin and 75 μg/ml streptomycin at 500×g for 5 minutes, and once in mTBM.The spermatozoa were diluted with mTBM to give a final concentration of 3×10 6 spermatozoa/ml.
After the IVM period, oocytes were briefly treated with 0.1% hyaluronidase in Dulbecco's phosphate-buffered saline (D-PBS, Gibco, USA) supplemented with 1 mg/ml BSA to remove cumulus cells, and were washed 2-3 times with modified Tris-buffered medium (mTBM) containing 1 mg/ml BSA and 2.5 mM caffeine sodium benzoate.After washing, groups of 25-30 oocytes were placed in 48-μl droplets of mTBM in 60-mm petri dishes that had been covered with warm mineral oil.Two microliters of spermatozoa suspension was added to each fertilization drop, resulting in a final concentration of 2.5×10 5 spermatozoa/ml.Oocytes and spermatozoa were coincubated for 6 h at 39°C and 5% CO 2 with maximum humidity.

In vitro culture
The presumptive zygotes (day 0) were washed three times with TL-HEPES solution, the impurities including sperm removed, and washed 2-3 times with PZM3 solution to which IGF-I and/or EGF (0, 1, 10 and 20 ng/ml) were supplemented.Then, the oocytes were inserted into 50 μl of PZM3 solution and in vitro development conducted by culturing at 39°C and 5% CO 2 in the culture medium.After 48 h of in vitro culture, the fertilization rate was examined.Then, 168 h after in vitro culture began, the embryo development of blastocysts and hatched blastocysts was examined.

Blastocyst differential staining
The zona pellucida of the blastocysts was removed with a 0.5% protease solution and washed 4-5 times in TL-HEPES solution with 0.1% PVA (TL-PVA).Zona-free blastocysts were incubated in a 1:5 dilution of rabbit antibovine whole serum in TL-PVA medium for 1 h.After being washed five more times in TL-PVA medium, blastocysts were re-incubated in a 1:10 dilution of a guinea pig complement in TL-PVA medium supplemented with 4 μg/ml propidium iodide (PI) and 4 μg/ml bisbenzimide for 1 h.The presumptive stained blastocysts were mounted on a slide and the cells were counted under a fluorescence microscope (Olympus, Tokyo, Japan).The bisbenzimidestained inner cell mass (ICM) nuclei fluoresced blue, and the trophectoderm (TE) nuclei, which were stained with both bisbenzimide and PI, fluoresced red or pink.

Statistical analysis
Data on embryo development were analyzed by the x 2test.All data for cell number were arcsine-transformed and analyzed by the General Linear Models Procedure with the Statistical Analysis System (SAS; Cary, USA).Treatment means were compared with Duncan's multiple range test; pvalues less than 0.05 were considered statistically significant.

Embryo development during IGF-I supplementation
The in vitro fertilization rates were 66.9%, 75.8%, 68.8% and 76.7% for the treatment groups, and there were no significant differences among the groups.The blastocyst development rates were 12.1%, 8.9%, 14.6% and 12.7% for the groups, and there were no significant differences among the groups.In addition, the blastocyst hatching rates were 5.3%, 7.1%, 21.7% and 5.3%, and again, there were no significant differences among the groups.

Embryo development during the EGF supplementation
The in vitro fertilization rates were 74.6%, 80.7%, 69.3% and 64.9% for the treatment groups, and the highest rate was that of the 1-ng/ml treatment group, which was significantly higher than the 10-ng/ml and 20-ng/ml treatment groups (p<0.05).In contrast, the blastocyst development rates were 16.7%, 13.2%, 8.8% and 7.2% for the treatment groups, and the highest rate was found in the non-supplemented group, where it was significantly higher than the 20-ng/ml treatment group (p<0.05).Also, the blastocyst hatching rates were 15.8%, 20.0%, 0.0% and 12.5%, and there were no significant differences among the groups.

Embryo development during the simultaneous supplementation of IGF-I and EGF
The in vitro fertilization rates were 67.6%, 69.2%, 76.9% and 63.5% for the treatment groups, and there were no significant differences among the groups.The blastocyst development rates were 13.5%, 10.9%, 12.2% and 13.5% for the treatment groups, and showed similar trends among the groups, while the blastocyst hatching rates were 15.0%, 17.6%, 10.5% and 9.5%, and there were no significant differences among the groups.

Number of cells
Figure 1 shows the number of cells of blastocysts produced under each condition.The total cell numbers were 20.5±4.2 ~ 22.4±4.9 for the treatment groups, and there were no significant differences among the groups.The ICM cell numbers ranged from 3.6±2.1 ~ 7.4±3.6,and there were no significant differences among the groups.Also, the TE cell numbers ranged from 13.5±2.4~ 17.1±4.5,and there were no significant differences among the groups.

DISCUSSION
In the various types of cells in the reproductive organs, diverse growth factors such as the IGF family, colony stimulating factor-I, transforming growth factor-β, leukemia inhibitor factor, granulocyte-macrophage colony-stimulating factor, EGF, and platelet-activating factor (Schell et al., 1994;Lonergan et al., 1996;Davies et al., 2004) are produced.In particular, the growth factor that is secreted from ovaries affects both nuclear and cytoplasmic maturation in the maturation process of oocytes (Motlik and Fulka, 1986), and the above growth factors play important roles in the development of blastocysts.Considering that the receptors for these growth factors appear on the cytoplasm surface, it was proved that as the embryo develops, these growth factors are necessary for the formation of blastocysts (Collier et al., 1990;Osterlund and Fried, 2000;Lee et al., 2005).
It was reported that among the above growth factors, IGF-I, which is a single chain polypeptide of 7.6 kDa that is structurally similar to proinsulin, is synthesized in the liver and fibroblast and found in follicular fluid (Spier and Echternkamp, 1995), uterine tubes (Schmidt et al., 1994), uterus (Geisert et al., 1991) and ovary, so as to affect the maturation of ova as well as embryo development (Watson et al., 1999), controlling the growth as well as the division of cells.Therefore, IGF-I supplementation to in vitro maturation and in vitro culture media of mouse (Doherty et al., 1994), rabbit (Herrler et al., 1998) and human (Spanos and Becker, 2000) oocysts enhanced embryo development by decreasing necrocytosis and increasing cell division (Devreker and Hardy, 1997).In previous studies, the amount of IGF-I supplementation during in vitro culture was 50-100 ng/ml in the case of cows (Byrne et al., 2002;Sirisathien et al., 2003) and 30-100 ng/ml (Desai et al., 2000;Fabian et al., 2004) for mice.The cell number of blastocysts which developed under each concentration was increased (mouse: Kurzawa et al., 2002;cow: Sirisathien et al., 2003;human: Spanos et al., 2000).Specifically, in porcines, the blastocyst formation rate as well as the cell number of blastocysts was increased (Lighten et al., 1997).However, in this study, there were no significant effects with regard to embryo development due to IGF-I supplementation of the in vitro culture medium of porcine embryos.
Among the growth factors in the reproductive organs, EGF, which is composed of 53 amino acids, plays a role as the cell division accelerator and also promotes growth of the epithelial cell, mesoderm cell, connective tissue, nerve cell and granule cell (Shiraga et al., 1997).In addition, it also exists on uterine tube cells, uterus cells and the oocyte cell surface (Kane et al., 1992) and it has been reported to be relevant to nuclear membrane disruption (Das et al., 1991), expansion of cumulus cells of immature bovine oocytes (Larson et al., 1990) and the cytoplasmic maturation of mouse, cow and human oocytes (Harper and Brackett, 1993;Park and Lin, 1993).The effects of EGF on the in vitro culture of oocytes (Wood and Kaye, 1989;Werb, 1990) have also been reviewed.In the case of mice, EGF promoted the formation of blastocoeles (Wood and Kaye, 1989;Paria et al., 1994) and increased blastocyst development rate of bovine embryos (Coskun et al., 1991;Harper and Brackett, 1993), as well as the cell number of blastocysts (Lee and Fukui, 1995) so as to increase embryo development rate (Wei et al., 2001).However, Abeydeera et al. (1998) reported that the supplementation of EGF to in vitro culture medium did not result in significant differences in blastocyst formation rate or cell numbers.
In this study, the supplementation of EGF to in vitro culture medium was not significantly effective and was in agreement with results of Abeydeera et al. (1998).When IGF-I and EGF were simultaneously supplemented, it was effective for the in vitro development of bovine (Rieger et al., 1998) and porcine (Yoon et al., 2003) embryos, but in this study the simultaneous supplementation of IGF-I and EGF to the in vitro culture medium did not affect blastocyst development rate or blastocyst hatching rate.
In terms of in vitro production of embryos, the differences in growth factors depending on animal species cannot be compared directly due to different culture conditions.However, it is considered that the effects of the growth factors on embryos were cancelled out by unknown factors due to the supplementation of protein sources such as BSA to the in vitro culture medium, PZM3.

Figure 1 .
Figure 1.Effects of IGF-I and EGF supplements on cell numbers of porcine blastocysts developed from in vitro-produced embryos.

Table 1 .
Effects of different concentrations of IGF-I supplement on the development of porcine in vitro-fertilized embryos

Table 2 .
Effects of different concentrations of EGF supplement on development of porcine in vitro-fertilized embryos a, b Within the same column, values with different superscripts differ significantly (p<0.05).

Table 3 .
Effects of simultaneous IGF-I and EGF supplements on development of porcine in vitro-fertilized embryos