Effect of addition of exogenous growth factor on in vitro development of preimplantation stage buffalo embryos

Effect of addition of exogenous growth factor on in vitro development of preimplantation stage buffalo embryos

CONTENTS 5. EFFECT OF ADDITION OF EXOGENOUS GROWTH FACTOR ON IN VITRO DEVELOPMENT OF PREIMPLANTATION STAGES BUFFALO EMBRYO Page 5.1 Introduction 5.2 Methods 5.3 Results 5.4 Discussions 75 77 79 81

INTRODUCTION 75 The early mammalian preimplantation embryos develop in the oviduct which secretes various proteins and growth factors (Gandolfi et a/., 1993). Manunalian embryos also secrete growth factors and have receptors for various growth factors (Rappolee et a/., 1988; Heyner et a/., 1989). It is therefore likely that growth factors influence embryonic development. Our studies reported in the previous chapter show that the highly sensitive reverse transcription-polymerase chain reaction can detect low abundance messenger RNA of growth factors at various preimplantation stages of buffalo embryos. It has been hypothesized that additional effects of various growth factors are likely to depend on the stage at which the embtyo is exposed to them. Many attempts have been made to investigate the effect of several growth factors on bovine and mouse embryonic development (Flood eta/., 1993; Larson et a/., 1992). Pari a and Dey, ( 1990) observed a cooperative interaction among preimplantation mouse embryos. Embryos cultured in groups showed better development to the blastocyst stage and higher cell numbers than embryos cultured singly. They speculated that embtyos produced growth factors which could act in an autocrine fashion to stimulate development. Insulin and insulin like growth factor-i has been shown to stimulate metabolism and growth of mouse embryos (Heyner et a/., 1989; Harvey and Kaye, 1988). There may be a synergistic effect of growth factors on embtyonic development since the oviduct secretes many growth promoting factors (Watson et a!., 1994). Some studies on the synergistic effect of growth factors such as PDGF and IGF-1 (Larson et a/., 1992), EGF and TGF-131 (Keefer, 1992), TGF-a and bovine fibroblast growth factor (Larson eta!., 1992) have been attempted in bovine embtyos. In order to analyze the biological activity of the growth factor ligands and

5. I lntrotluction 76 receptors detected in buffalo embryos and oviduct cells, the effect of insulin and IGF-1 growth factors m culture medium on buffalo embryo development was investigated.

METHODS 77 Buffalo ovaries obtained from an abattoir were transported to the laboratory in saline supplemented with 50 mg/ml gentamicin and were maintained at 30 to 350C. The ovaries were rinsed thrice in fresh saline within 3-4 h of collection. The detailed culture procedures employed in the production of preimplantation buffalo embryos are outlined in the protocols section. In vitro oocyte maturation Immature oocytes were aspirated from follicles of about 2-6 mm diameter. These oocytes were washed thrice with TL-Hepes, twice with maturation medium and transfened to I 00 1-11 droplets of maturation medium (20 oocytes per drop) and cultured for 24 hat 390C in a moist atmosphere of 5% C02. In vitro fertilization Frozen thawed buffalo semen was used for in vitro fertilization. Matured oocytes were transfened to 50 1-11 droplets of fertilization medium (I 0 oocytes per drop). Live spermatozoa were separated using the percoll gradient method. Spermatozoa were counted in a haemocytometer and 2 1-11 of sperm suspension having a final concentration of 2x I o6 spetmatozoa I ml was added to each 50 1-11 droplet. Spetm and oocytes were then co-incubated at 390C in 5% C02 in a moist atmosphere for 24 h. Embryo culture After fettilization, the cumulus cells of presumptive zygotes were removed with repeated pipetting and embryos were washed in CR I aa medium. Experiment I: Embryos were transfeited to I 00 1-11 droplets of CR I aa medium (20 embryos per drop) containing I, 10, and 100 ng/ml IGF-1 and co-cultured with

5.2 Methotl.'i 78 buffalo oviductal epithelial cells. Cultures were incubated at 390C m an atmosphere of 5% C02 for 8 to 9 days. Experiment 2: Embryos were transferred to I 00 J.ll droplets of CR I aa medium (20 embryos per drop) containing I, I 0, and I 00 ng/ml insulin and co-cultured with buffalo oviductal epithelial cells. Cultures were incubated at 390C in an atmosphere of 5% C02 for 8 to 9 days. Statistical analysis The raw data was analyzed using the chi square (2x2) test. A "p" value of <0.05 was considered significant:

RESULTS 79 Experiment 1: Effect of various doses of IGF-1 (I, 10 and 100 ng) in the culture medium was determined. Addition of IGF-1 at various concentrations (I, 10, and 100 nglml) did not increase the cleavage rate which was found to be 75.5, 62.2 and 74.7 % respectively over that of control culture medium where no IGF-1 was added (74.4 %). Addition of I and 10 ng IGF-1 increased development rates to blastocyst (26.9 and 22.9 % respectively) as compared to the control culture medium (I 0.4 %). However, there was no significant difference within these treatments (Table 4). Addition of 100 ng IGF-1, increased rate of formation of blastocyst (32.3 %) when compared to embryos grown in the control medium (P < 0.05). Experiment 2: The media containing various doses of insulin (I, I 0, and I 00 J.tg) showed that the development rate of blastocysts were significantly increased (32.1 %) when l J.tg/ml insulin was added to CR l aa medium in comparison to the control culture medium (P<O.Ol). However, increasing the concentration of insulin to 10 and l 00 nglml significantly reduced the rate of blastocyst development (Table 5).

5.3 Results 80 Table 4. Development of IVMIIVF buffalo zygotes co-cultured with BOEC in CRlaa medium supplemented with IGF-1 Treatment No. of Oocytes No. of Zygotes No. of Blastocyst cultured cleaved (%)} developed ( 0 /o) CRlaa 180 134 (74.4) 14 (l0.4)a CRiaa + lng IGF-1 172 130 (75.5) 35 (26.9) CR laa + 10 ng IGF-J 187 122 (62.2) 28 (22.9) CRiaa + 100 ng IGF-1 190 142 (74.7) 46 (32.3)b a, b Values with different superscript letter are significantly different (P < 0.05). Table 5. Development of IVM/IVF buffalo zygotes co-cultured with BOEC in CRl aa medium supplemented with insulin. Treatment No. of Oocytes No. of Zygotes No. of Blastocyst cultured cleaved (%) developed (%) CRiaa 180 134 (74.4) 14 (10.4)a CR 1 aa + lj.lg insulin 120 80 (66.6) 26 (32.1 )b CR I aa + I Oj.lg insulin 90 60 (66.6) 8 (13.3) CR I aa + I OOj.lg insulin 70 40 (57.1) 2 ( 5.0) a, b Values with different superscript letter are significantly different (P < 0.0 I).

DISCUSSION 81 Insulin and insulin-like growth factor-1 are small mitogenic polypeptides with structural and functional homologies to each other. In addition to their broad range of effects on a number of cells, they have been demonstrated to be expressed in a developmentally regulated manner during preimplantation embryo development (Schultz and Heyner, 1993). The insulin family of growth factors exert this biological action by binding to specific cell surface glycoprotein receptors. Based on observations regarding the localization of the IGF-1, IGF-11 and insulin receptors which are preferentially localized in almost all if not in certain stages from the 2-cell to the blastocyst stage of preimplantation buffalo embryos, the development media co-cultured with BOEC was supplemented with different concentrations of IGF-1 or insulin. The results demonstrate that the development of embryos into blastocysts were significantly increased with IGF-1 at a high concentrations and with insulin at lower concentrations. A similar observation was also made when IGF-1 at a high dose affected cellular proliferation in mouse embryos (Rappolee eta/., 1992) and addition of insulin at a low dose (40 ng/ml) to the culture medium stimulated protein synthesis and cellular proliferation in preimplantation mouse embryos (Heyner et a/., 1989; Rao et a/., 1990). Studies have shown that the addition of insulin or insulin-like growth factors to medium for culture of preimplantation mammalian embryos have a stimulatory effect on embryo metabolism and cellular proliferation in developing embtyos of mouse (Harvey and Kaye, 1988; Heyner eta/., 1989; Gardner and Kaye, 1991; Hogan et a/., 1991; Kaye eta/., 1992), rat (Zhang and Atmstrong, 1990), cow (Zhang eta/., 1991) and pig (Lewis eta/., 1992). However Lee and Fukui ( 1995) did not get any improvement in bovine embryonic development although, there have been repm1s

.l. 4 Di.'lcU.'I.fiiion 82 that IGF-1 and its receptor gene are present in cattle embryos. The dose of IGF-1 used may not have been optimal for stimulation of embryonic development in this study. IGF-1 receptor is not functional in pig embryos (Rappolee eta/., 1990). Many growth factors have both growth-promoting and growth-inhibiting activities, depending on the biological context. Cellular response to exogenous growth factors in vitro is dose dependent and it varies in different species. For example, Harvey and Kaye ( 1992) have demonstrated that a concentration of IGF-1 as low as 0.1 to 1.0 ng/ml results in cellular proliferation of mouse embryos. The total cell number (TCN), trophectodetm (TE) and inner cell mass (ICM) significantly improved following in vitro culture in medium supplemented with JGF-1 in buffalo (Namla et a/., 1996). As reported earlier in mouse, IGF-1 increases the total number of cells in blastocysts by increasing the ICM (Harvey and Kaye, 1992; Smith et a/., 1993), whereas in buffalo, TE increase was more pronounced than ICM with addition of IGF-1 (Namla eta/., 1996). Although the levels of IGF-1 present in the reproductive tract of mouse is not known, but recent analysis indicates that there is about 30 ng/ml IGF-1 in porcine oviductal fluid. It has been also demonstrated that the cleavage rate of pig oocytes was markedly increased in a dose dependant manner by addition of IGF-1 during oocyte maturation (Xia eta/., 1994). Oviductal cells also play a specific role in the stimulation of early development, perhaps through the secretion of undefined embtyotrophic factors into the culture medium (Eyeston and First, 1989; Boice eta/., 1990; Ellington et a/., 1990; Wiemer eta/., 1991). Control of growth factors and differentiation of mammalian embtyogenesis may be regulated by growth factors from embryo or matemal sources. Thus the oviductal cells may be responsible for activation of

83 specific genes which initiate autonomous embryonic development (Caird and Rexroad, 1989). Rosenkrans ( 1990) has reported the development of bovine embryos to the morula and blastocyst stage in CR l aa medium without co-culture with oviductal cells. Zhang et a/., (1992) demonstrated that the development of bovine embryos to the blastocyst stage is increased when embryos were cultured in CR I aa medium and co-cultured with oviductal cells. However, reports indicate that the developmental rates of bovine embtyos resulting from in vitro co-culture are usually slower than that of normal in vivo embryos and that is also a reduction in inner cell mass in blastocysts produced in vitro. Addition of TGF-a to embryo co-culture system with oviductal cells is repmted to enhance blastocyst formation (Marquent-Le Guienne e1 a/., 1989). Thibodeaux et a/. (1993) has demonstrated that the culture of bovine embtyos in oviductal cells supplemented with PDGF increases the rate of development to the blastocyst stage. Therefore, addition of exogenous growth factors at a physiological level after localization of specific growth factor ligands and receptors on preimplantation embryos and oviductal cells, perhaps, increase the developmental rate of embryos in buffalo.