Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos

Efeito do fator de crescimento semelhante à insulina-1 durante a maturação in vitro dos oócitos e cultivo in vitro de embriões bovinos

Abstracts

The effects of insulin-like growth factor-I (IGF-I) on in vitro maturation (IVM) (experiment I) and on in vitro embryo development (experiment II) of bovine oocytes fertilized in vitro, were evaluated in terms of cleavage (CR), blastocyst (BR) and hatching (HR) rates. For IVM, immature cumulus-oocyte complexes were cultured in TCM-199 medium supplemented with Hepes, sodium bicarbonate, sodium pyruvate, additives, fetal calf serum (B-199 medium) and gonadotropins (14 U/ml PMSG and 7 U/ml hCG). For embryo development, the oocytes/zygotes were cultured in B-199 medium with bovine oviduct epithelial cells in suspension under silicon oil. Treatments for in vitro culture conditions for both experiments were: 1- B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. All cultures were performed at 38.5ºC in 5% CO2 in air and the data were analyzed by chi-square test. In experiment I, there were no differences (P>0.05) among treatments for CR, BR or HR. In experiment II, the addition of IGF-I to the embryo culture medium (ECM) resulted in a significant increase in CR while for BR and HR this effect was not observed. The addition of 200 ng/ml IGF-I to ECM increased CR (71.1%) when compared to 100 ng/ml IGF-I (57.6%) or control (56.7%) groups, however, there were no differences when compared to 50 (69.4%) or 10 ng/ml (73.1%) groups. There was no beneficial effect of the addition of IGF-I in the IVM or ECM media on the in vitro development of embryos produced from oocytes matured and fertilized in vitro.

Bovine; cleavage rate; IGF-I; in vitro maturation; in vitro fertilization

Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos

[Efeito do fator de crescimento semelhante à insulina-1 durante a maturação in vitro dos oócitos e cultivo in vitro de embriões bovinos]

M.D. Quetglas¹, L.A. Coelho^2*, J.M. Garcia¹, E.B. Oliveira Filho¹, C.R. Esper¹

¹Faculdade de Ciências Agrárias e Veterinárias - UNESP, Jaboticabal

2Faculdade de Zootecnia e Engenharia de Alimentos da USP

Av. Duque de Caxias Norte, 225

13630-000 - Pirassununga, SP

Recebido para publicação, após modificações, em 29 de novembro de 2000.

E-mail: [e-mail]

ABSTRACT

The effects of insulin-like growth factor-I (IGF-I) on in vitro maturation (IVM) (experiment I) and on in vitro embryo development (experiment II) of bovine oocytes fertilized in vitro, were evaluated in terms of cleavage (CR), blastocyst (BR) and hatching (HR) rates. For IVM, immature cumulus-oocyte complexes were cultured in TCM-199 medium supplemented with Hepes, sodium bicarbonate, sodium pyruvate, additives, fetal calf serum (B-199 medium) and gonadotropins (14 U/ml PMSG and 7 U/ml hCG). For embryo development, the oocytes/zygotes were cultured in B-199 medium with bovine oviduct epithelial cells in suspension under silicon oil. Treatments for in vitro culture conditions for both experiments were: 1- B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. All cultures were performed at 38.5^oC in 5% CO₂ in air and the data were analyzed by chi-square test. In experiment I, there were no differences (P>0.05) among treatments for CR, BR or HR. In experiment II, the addition of IGF-I to the embryo culture medium (ECM) resulted in a significant increase in CR while for BR and HR this effect was not observed. The addition of 200 ng/ml IGF-I to ECM increased CR (71.1%) when compared to 100 ng/ml IGF-I (57.6%) or control (56.7%) groups, however, there were no differences when compared to 50 (69.4%) or 10 ng/ml (73.1%) groups. There was no beneficial effect of the addition of IGF-I in the IVM or ECM media on the in vitro development of embryos produced from oocytes matured and fertilized in vitro.

Keywords: Bovine, cleavage rate, IGF-I, in vitro maturation, in vitro fertilization

RESUMO

Avaliaram-se o efeito do IGF-I na maturação in vitro (MIV) (experimento I) e no desenvolvimento embrionário (DE) (experimento II) de oócitos bovinos fecundados in vitro, quanto às taxas de clivagem (TC), de blastocistos (TB) e de eclosão (TE). Para MIV, complexos cumulus-oócitos imaturos foram cultivados em meio TCM-199 suplementado com HEPES, bicarbonato e piruvato de sódio, aditivos, soro fetal bovino (meio B-199) e gonadotrofinas 14U/ml de PMSG e 7U/ml de hCG). Para o desenvolvimento embrionário, os oócitos/zigotos foram cultivados em meio B-199 com células epiteliais do oviduto bovino em suspensão sob óleo de silicone. As condições de cultivo in vitro para ambos os experimentos seguiram os tratamentos: 1- meio B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. Todas as culturas foram realizadas a 38,5° C em atmosfera com 5% de CO₂ e os dados foram analisados pelo teste do qui-quadrado. No experimento I, não houve diferença (P>0,05) entre os tratamentos quanto às TC, TB e TE, quando o meio de MIV foi suplementado com IGF-I. No experimento II, a adição de IGF-I ao meio de DE resultou em aumento na TC (P<0,05) mas não influenciou a TB e a TE. A adição de 200 ng/ml de IGF-I ao meio DE melhorou a TC (71,1%) quando comparada com a TC dos grupos de 100 ng/ml de IGF-I (57,6%) ou controle (56,7%), entretanto não houve diferença quando comparada com a dos grupos de 50 ng/ml (69,4%) ou 10 ng/ml (73,1%) de IGF-I. Não houve efeito benéfico na adição de 10 a 200 ng/ml de IGF-I nos meios de MIV e de DE com relação ao desenvolvimento de embriões produzidos a partir de oócitos maturados e fecundados in vitro.

Palavras-chave: Bovino, fecundação in vitro, IGF-I, maturação in vitro, taxa de clivagem

INTRODUCTION

Successful methods have been developed for producing bovine embryos in vitro (IVP). However, the percentage of blastocysts and hatched blastocysts obtained from IVP systems is extremely variable (Looney et al., 1994; Elmileik et al., 1995; Jaakma et al., 1997; Choi et al., 1998). The development of an efficient culture system, which can support the embryonic development of oocytes following IVM/IVF resulting in a high proportion of viable morulae and blastocysts would be beneficial to both research and commercial efforts (Keskintepe & Brackett, 1996).

Growth factors have been shown to play a regulatory role in the functioning of the ovary (Echternkamp et al., 1994) and of the uterus (Boehm et al., 1990), resulting in a trophic effect on the endometrium and embryo. Insulin-like growth factor I (IGF-I) is an important growth factor in the blood and is mainly responsible for the interaction between the uterine endometrium and conceptus (Simmen et al., 1993). Several research groups reported that addition of IGF-I to culture media stimulates the maturation of oocytes and development of murine (Harvey & Kaye, 1992), bovine (Herrler et al., 1993; Lorenzo et al., 1994; Matsui et al., 1997), porcine (Xia et al., 1994) and human (Lighten et al., 1998) embryos in vitro.

The purpose of this experiment was to assess whether in vitro maturation (IVM) and embryo culture media supplemented with IGF-I could improve embryo development and to establish which concentration of IGF-I could be used on in vitro embryo production systems.

MATERIALS AND METHODS

Immature cumulus-oocyte complexes (COCs) were aspirated from visible ovarian follicles from slaughtered cows and were washed twice in TCM-199 medium (Sigma, St. Louis, MO, USA) containing additives, 25mM Hepes¹, 75µg/ml kanamycin sulfate¹ (H-199 medium) and 10% fetal calf serum (FCS) and once in maturation medium. The basic maturation medium (B-199) was made up by TCM-199 medium supplemented with Hepes¹ (16.79mM), sodium bicarbonate¹ (28.57mM), sodium pyruvate¹ (2.73mM) and FCS (10%). For IVM, the oocytes were cultured in B-199 medium supplemented with 14 U/ml pregnant mare serum gonadotropin¹ (PMSG) and 7 U/ml human chorionic gonadotropin (hCG, Serono, Rome, Italy) (hCG) for 22 to 24h.

For insemination, 0.50ml straws of frozen semen from the same bull were thawed and separated by discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient (Avery & Greve, 1995). The sperm concentration was adjusted to 10 ´ 10⁶ cells/ml and capacitation was induced with 10 µg/ml heparin¹. After IVM, the oocytes were washed twice in serum-free H-199 medium, once in fertilization (IVF-TALP) medium (Coelho, 1993), and transferred to 100µl droplets (25 oocytes per droplet) of the sperm suspension under silicon oil. Oocytes and spermatozoa were co-incubated for 48h and after that the oocytes/zygotes were transferred into droplets prepared with embryo culture medium which consisted of 90µl B-199 medium and 2µl bovine oviduct epithelial cells (BOEC) in suspension under silicon oil. They were further cultured for nine days. All the cultures were performed at 38.5ºC in 5% CO₂ in air. The blastocyst (BR) and hatching (HR) rates were evaluated on days seven and nine post insemination, respectively.

The effects of the addition of IGF-I on IVM and on embryo culture media were evaluated on experiments I and II, respectively. Treatments for in vitro culture conditions for both experiments were: 1- B-199 + 200 ng/ml IGF-I (B200); 2- B-199 + 100ng/ml IGF-I (B100); 3- B-199 + 50ng/ml IGF-I (B50); 4- B-199 + 10ng/ml IGF-I (B10); 5- B-199 + 0 ng/ml IGF-I (control). The oocytes used in each treatment were 141, 148, 92, 68, 116 and 201, 144, 196, 175, 147 for treatment 1, 2, 3, 4 and 5, respectively, in experiments I and II. The CR, BR and HR were analyzed by chi-square test (c²).

RESULTS AND DISCUSSION

In experiment I, there were no differences (P>0.05) among treatments regarding cleavage rate, embryo development to morula/blastocysts stages or to the hatching of blastocysts when the IVM medium was supplemented with IGF-I (Table 1). These data are in agreement with those of others which showed that IGF-I, if included in IVM medium, had no effect on development of bovine embryos after IVF (Herrler et al., 1993). However, it has been reported that the CR of porcine oocytes was markedly stimulated in a dose-dependent manner by addition of IGF-I to the IVM medium (Xia et al., 1994). The increase of CR was the greatest at 50ng/ml IGF-I but this effect was evident only when the IVM medium contained no gonadotropins. These findings suggest that IGF-I treatment during IVM was more effective than the gonadotropins in enhancing subsequent embryo development and the combination of both of them caused no further enhancement of embryo development. The independent effect of IGF-I was not examined, however, the present results are in agreement with those from the study of Xia et al. (1994), in which no effect of IGF-I on IVM of oocytes has been demonstrated, when gonadotropins were present in the medium.

In experiment II, the addition of IGF-I to the embryo culture medium did result in a significant increase in the percentage of oocytes cleaved while the blastocyst or hatched rates did not so (Table 2). The addition of 200 ng/ml IGF-I to the embryo culture medium improved the CR (71.1%) compared to 100 ng/ml IGF-I (57.6%) or control (56.7%) groups, however, there were no differences when compared to 50 ng/ml (69.4%) or 10 ng/ml (73.1%) IGF-I groups (Table 2). This difference on the CR may have been only a temporary effect, as no positive effect of IGF-I on BR and on HR was detected. These data were consistent with the results from studies that showed no beneficial effect of IGF-I on bovine embryonic development (Herrler et al., 1993; Lee & Fukui, 1995) but conflict with results of studies reporting its positive effect (Matsui et al., 1997; Palma et al., 1997). These divergent observations may be due to different culture conditions. In this study, the embryo culture medium was supplemented with bovine oviduct epithelial cells (BOEC) and fetal calf serum, which may influence embryonic development. The BOEC culture systems are lacking adequate definition to assure quality control and repeatability and serum contains growth factors that may stimulate the embryonic development (Keskintepe & Brackett, 1996). Therefore, it is difficult to interpret the effects of a specific substance on embryo development when media containing BOEC or serum are used. It has been shown that, even when chemically defined culture systems are used, the addition of 1 ng/ml IGF-I on embryo culture had no positive effect on embryonic development (Herrler et al., 1993). However, stimulatory effects on the development of bovine embryos were observed within the range of 2 to 200 ng/ml when these amino acids were present in the culture medium indicating that the supplementation of amino acids is necessary for IGF-I to exert its beneficial effect (Matsui et al., 1997). In the present study, the concentration of IGF-I ranging from 10 to 200 ng/ml was not sufficient to show the beneficial effect on embryo development probably due to the lack of the ideal amino acid concentrations in the embryo medium and the culture conditions not being chemically defined.

Finally, in the described culture system, the addition of IGF-I in the in vitro maturation or embryo culture media had no beneficial effect on the in vitro development of bovine embryos produced from oocytes matured and fertilized in vitro.

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Publication Dates

• Publication in this collection
  05 June 2002
• Date of issue
  Apr 2001

History

• Received
  29 Nov 2000