Vet Res Commun (2010) 34:11–18 DOI 10.1007/s11259-009-9325-1 ORIGINAL ARTICLE
Effect of nerve growth factor (NGF) on the development of preimplantation rabbit embryos in vitro Yijin Pei
Accepted: 2 October 2009 / Published online: 16 October 2009 # Springer Science + Business Media B.V. 2009
Abstract This study aimed to investigate the effect of nerve growth factor (NGF) on the development of preimplantation rabbit embryos in vitro. Zygotes were collected from superovulated New Zealand rabbits 19 h after injection of hCG and immediately mating and cultured in TCM-199 plus fatty-acid free BSA with different concentrations of NGF. Zygotes not treated with NGF served as control. At 24 h, 48 h, 72 h and 96 h of the culture, the numbers of the early cleavage stage, morulae, blastocysts and hatching blastocysts were determined. The intrazonal diameter of the blastocyst and the total cell numbers per blastocyst were measured after 96 h of culture. The results showed: (1) NGF at 100 ng/mL and 1000 ng/mL could improve the numbers of the hatching blastocysts which developed compared to the control treatment (p<0.05); (2) All concentrations of NGF increased the total cell numbers in the blastocysts compared to the control treatment (p<0.05); (3) NGF had no significant effect on the blastocyst intrazonal diameter of the blastocysts at 96 h of culture (p=0.493); (4) The proportion in the early cleavage stage at 24 h of culture (p= 0.635), of morulae at 48 h of culture (p=0.812) and of blastocysts at 72 h of culture (p= 0.812) in all treatments were not significantly different. Keywords Development . Nerve growth factor . Rabbit . Zygotes Abbreviations BSA bovine serum albumin EGF epiderminal growth factor HB-EGF heparin-binding epiderminal growth factor hCG human chorionic gonadotropin
This study was supported by the Doctoral Scientific Research Foundation of Guangdong Medical College. Y. Pei Department of Physiology, Guangdong Medical College, Zhanjiang, Guangdong, People’s Republic of China Y. Pei (*) No.1 Xincheng Road, Songshan Lake, Dongguan, Guangdong 523808, People’s Republic China e-mail:
[email protected]
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LIF NGF TGF
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leukemia inhibitory factor nerve growth factor transforming growth factor
Introduction The in vitro development of the preimplantation embryos was not equivalent to that which occurs in vivo, as manifested by reduced cell numbers, consistent with a progressive loss of viability, reduced metabolism and altered imprinted genes (for a review, see Thompson 2000; Corcoran et al. 2005), indicating that the culture conditions were sub-optimal and suggesting that maternal factors may be involved in regulating normal development. Many investigators (Schini and Bavister 1988; Desai et al. 2000; Vireque et al. 2009) have tried to improve the culture system and to replicate the in vivo environment in order to enhance in vitro development and to understand the role of growth factors in development. There is considerable evidence that peptide growth factors play a role in the development of preimplantation embryos in a variety of mammalian species (for a review, see Richter 2008). Culture media supplemented with various growth factors have been used to study their possible effect on in vitro development. A number of studies have reported that for in vitro culture of preimplantation embryos, the addition of exogenous growth factors, including the epidermal growth factor (EGF) family, the insulin and insulin-like family of growth factors, the transforming growth factor-β (TGF-β) family, the platelet-derived growth factor and fibroblast growth factor family, could affect the preimplantation development and result in a broad range of effects that include stimulation of protein synthesis in mice (Wood and Kaye 1989) and pigs (Lewis et al. 1992), promotion of blastocyst formation in cattle (Larson et al. 1992; Yang et al. 1993; Lima et al. 2006) and mice (Paria and Dey 1990), increasing the percentage of hatching blastocysts in mice (Lim et al. 2006) and humans (Martin et al. 1998), increase in the number cells in blastocysts in mice (Lin et al. 2003) and cattle (Sirisathien et al. 2003), and prevention of apoptosis in rabbits (Herrler et al. 1998) and human (Spanos et al. 2000).Clearly a better understanding of the role of growth factors in development could help to elucidate the mechanisms controlling embryonic development and to improve the viability and development of embryos in vitro. Nerve growth factor (NGF) has been shown to mediate cell growth and differentiation of target cells, including neurons and non-neuronal cells, and to have effects on neurogenesis and morphogenesis during rat embryonic and fetal life (Katz et al. 1990; Wheeler and Bothwell 1992; Miralles et al. 1998).In the reproductive tract, NGF may participate in fertilization mechanisms by cytoskeletal-mediated activation of spermatozoa locomotion, much in the same way as in neurite outgrowth, or by favoring egg implantation by inhibition of rejection through the immune system (Levi-Montalcini 1987). As it is expressed in the pregnant murine uterus, maternally derived NGF may play a role in placentation in mice by promoting giant-cell transformation of trophoblastic cells through p75 NGFR (Kanai-Azuma et al. 1997). NGF mRNA was detected in rat later embryonic and fetal development (Varol et al. 2000). There is evidence that NGF treatment induced a marked cumulus expansion and a progressive cumulus-oocyte uncoupling, and promoted ovine oocyte GVBD (Barboni et al. 2002).Recent evidence has shown that NGF also has effects on the proliferation or differentiation of pluripotent murine and human embryonic stem cells, derived from preimplantation embryos (Schuldiner et al. 2000; Moscatelli et al. 2009). Thus, NGF seems to have multiple important functions in embryonic and fetal development.
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However, little is known about the impact of NGF on early embryonic development. This study was therefore designed to investigate whether NGF has any effect on the in vitro development of rabbit zygotes to the hatching blastocyst stage.
Materials and methods Superovulation and recovery of embryos Sexually mature, New Zealand white, female rabbits, weighing 3.5 to 4 kg, were superovulated by injection of 20 IU/kg pregnant mare serum gonadotropin (Ningbo Hormone Product Co.Ltd., Ningbo, China), followed 72 h later by the administration of 160 IU human chorionic gonadotropin (hCG, Serono Laboratories, Aubonne, Switzerland), as described previously (Chrenek et al. 1998), and mated with fertile male rabbits from the same breed immediately after the hCG injection. Zygotes were collected from the oviducts 19 h after the administration of the hCG, by flushing with phosphate-buffered saline containing 1 mg/mL polyvinyl alcohol (P-8136, Sigma Chemical Co., St Louis, MO, USA). Only selected embryos at exactly the zygote stage were used. Embryos with irregular shape were considered abnormal and discarded (Carney and Foote 1991). Embryo culture Normal embryos from each donor were distributed equally and randomly into control groups and into groups receiving various concentrations of NGF, as follows: (1)TCM199 containing Earle’s salts (Gibco Life Technologies, GrandIsland, NY,USA)+1.5% fattyacid free BSA (Sigma),control group; (2) TCM199+1.5%BSA+1 ng/mL NGF (From mouse submaxillary glands, Sigma); (3) TCM199+1.5%BSA+10 ng/mL NGF; (4) TCM199+1.5%BSA+100 ng/mL NGF; (5) TCM199+1.5%BSA+1000 ng/mL NGF. Embryos were cultured under mineral oil (M-8410, Sigma) at 39°C under 5% CO2 with saturated humidity in 50 μl droplets in a 60×15 mm Falcon sterile petri dish. The media were replaced with fresh, equilibrated medium after 48 h.The experiment was replicated 7 times. At 24 h, 48 h, 72 h and 96 h into the culture, the embryos were evaluated as early cleavage-stage embryos, morulae, blastocysts or hatched blastocysts, respectively. After 96 h of the culture the intrazonal blastocyst diameters were quickly measured using a microscope fitted with a calibrated eyepiece micrometer, as described by Hallden and colleagues (1992). Immediately after this measurement, the blastocysts were stained with Hoechst 33342 dye (Sigma), as described previously (Pursel et al. 1985, as modified by Yang et al. 1990), and the number of total cells per blastocyst were counted using a Nikon inverted microscope equipped with an ultraviolet light source and a ×40 fluorescence objective (Nikon Inc., Melville, NY, USA). Statistical analysis The percentages of zygotes developing to the various morphological stages were subjected to arcsin (square root) transformation and analyzed by ANOVA, followed by Fisher’s protected least significant difference (LSD) test. Data on the cell numbers in the blastocysts and the intrazonal diameter of blastocysts are presented as the mean±SD and similarly analyzed. Differences were considered statistically significant at P<0.05.
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Table 1 Culture of rabbit zygotes in TCM-199 medium with various NGF concentrations No.of morula No.of 2- to 16-cell embryos (%)a (48 h) a (%) (24 h)
No.of blastocyst (%) a (72 h)
No. of hatching blastocyst (%) (96 h)
40(63.5)
31(49.2)
20(31.8)b
57(98.3)
42(72.4)
33(56.9)
22(37.9)bc
56
54(96.4)
46(82.1)
31(55.4)
22(39.3)bc
58 63
57(98.3) 62(98.4)
44(75.9) 52(82.5)
36(62.1) 42(66.7)
28(48.3)c 39(61.9)d
Treatment
No.of zygotes
1(control)
63
58(92.1)
2(1 ng/mL NGF)
58
3(10 ng/mL NGF 4(100 ng/mL NGF) 5(1000 ng/mL NGF)
a No significant differences between the five groups (p>0.05) for the 2- to 16-cell stage at 24 h of culture, morulae at 48 h of culture or blastocyst at 72 h of culture.
Numbers with different superscripts within the column for hatching blastocysts at 96 h of culture are significantly different (p<0.05 in each case).
Results The effect of NGF on the proportions of the various embryonic development stages are shown in Table 1. NGF at 100 ng/mL (p=0.001<0.05) and 1000 ng/mL (p=0.035<0.05) improved the rate of the formation of hatching blastocysts compared to the control group. The proportions of cleavage stage (p=0.635>0.05 for all), morula (p=0.812>0.05 for all), blastocyst (p=0.650>0.05 for all) for all the groups were not significantly different. The effect of NGF on blastocyst cell number at 96 h culture is shown in Table 2 and the effect of NGF on the blastocyst intrazonal diameter is shown in Table 3. All concentrations of NGF significantly increased the total cell number in the blastocysts compared to the control (p< 0.05). None of the concentrations of NGF affected the blastocyst intrazonal diameter at 96 h of the culture (p=0.493>0.05 for all).
Discussion TCM-199 plus BSA was adopted as the control culture medium, as it was used in earlier cultures of rabbit embryos (Carney and Foote 1991) when it was supplemented with various growth factors, including EGF, insulin, transferrin and selenium. The present study was performed in the absence of such proteins other than BSA, in order to more clearly evaluate the effect of NGF. BSA is regarded as a rather variable factor, different lots of BSA varying Table 2 The effect of NGF on the total cell numbers in the blastocysts over 96 h culture in medium 199 Treatment
No.of blastocyst
Total cell number
1 (control)
20
117±6a
2 (1 ng/mL NGF) 3 (10 ng/mL)
20 20
145±16b 147±10b
4 (100 ng/mL)
20
169±11c
5 (1000 ng/mL)
20
177±6c
Data are presented as mean±SD. Numbers with different superscripts within the column for the total cell number in the blastocysts at 96 h of culture are significantly different (p<0.05 in each case).
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Table 3 The effect of various concentrations of NGF on intrazonal diameter of the blastocyst developed from rabbit zygotes in medium 199 for 96 h culture Treatment
No.of blastocyst
Intrazonal diameter (μm)
1(control)
19
159±16
2(1 ng/mL NGF)
19
166±13
3(10 ng/mL)
19
161±13
4(100 ng/mL)
21
163±14
5(1000 ng/mL)
22
165±10
Data are presented as mean±SD. No significant differences were observed between five groups (p>0.05).
greatly in their growth-promoting effect (Kane 1983).To eliminate this variability, the same batch of fatty acid-free BSA was used throughout this study. In the present study, without the addition of NGF, the rates of development from 2-to 16 cell stage at 24 h, to morula at 48 h, to blastocysts at 72 h,or to hatching blastocysts at 96 h of culture were 92.1%,63.5%,49.2% and 31.8%, respectively, which demonstrated that a proportion of rabbit 1-cell embryos can develop to hatching blastocysts in the semi-defined culture medium. There have been a few reports on the effects of some growth factors and cytokines on the in vitro development of rabbit embryos. Insulin and insulin-like growth factor-1 were reported to promote development of rabbit blastocysts (Herrler et al. 1998), while the addition of platelet-derived growth factor, mouse leukemia inhibitory factor (LIF) or human LIF did not result in detectable difference in the total cell number in rabbit blastocysts (Giles and Foote 1997). NGF has now been clearly shown to have positive effects on the development of rabbit embryos in a semi-defined culture medium. 100 ng/mL or 1000 ng/mL NGF increased rabbit embryo hatching after 96 h of culture in a semi-defined culture medium to 48.3% or 61.9% respectively, compared with 31.8% in the control (p<0.05). NGF appeared to increase the percentages of hatching blastocysts in a concentration-dependent manner. Also the higher concentrations of NGF or and 1000 ng/mL were associated with a gather increase in the mean total cell numbers in the blastocyst. EGF (Desai et al. 2000) and HB-EGF (Martin et al. 1998; Lim et al. 2006) have also been reported to stimulate the proportion of hatching blastocysts in several mammalian species. The mechanisms by which growth factors promote the formation of hatching blastocysts are not well understood. The increased rate of zona hatching caused by EGF could be due to increased plasminogen activator activity of the trophectoderm cells by this growth factor (Galway et al. 1989; Paria and Dey 1990), while the beneficial effect of HBEGF on hatching suggests possibly by stimulating the production of an embryo-derived factor involved in thinning of the zona or promoting blastocoele expansion (Martin et al. 1998).The effect of NGF in promoting the proportion of hatching blastocysts may be due to the increase in cell numbers. After 96 h in culture, NGF had no effect on the intrazonal diameter of blastocysts. However cell counts are considered to be a more useful indicator of embryonic development than diameter (Hallden et al. 1992). Low number of cells an embryo has adverse effect on the differentiation and viability of the embryos (Edwards 1995). Supplementing the medium with NGF from the zygotes stage increased the total cell number for blastocysts at all concentrations of NGF (p<0.05). The addition of EGF or
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TGF-α (Paria and Dey 1990) in mice to culture media for embryos has also been shown to promote the cell numbers in blastocysts. Increases in cell number per blastocyst induced by EGF or TGF-α may reflect the mitogenic effect on preimplantation development (Paria and Dey 1990). The effect of NGF on cell number for blastocysts suggested that NGF may play a role on the cell division of rabbit embryos and so may affect further development. In the current study there was not beneficial or inhibitory effect of NGF on embryonic development from 2-to 16 cell stage at 24 h of culture, to morula at 48 h of culture, or to blastocysts at 72 h of culture. LIF was also reported to have no obvious effect on the early murine embryos (2 to 16 cell stage) but to have a positive effect on blastocyst formation (Tsai et al. 2000).This may result from the different requirements during different embryo development, since the requirements for growth factors and nutrition increased as the embryos grow (Tsai et al. 2000). Although our present study has shown that NGF has beneficial effects on the rabbit embryonic development in vitro, the mechanisms by which NGF exerts such actions are still unclear. While it has been reported that maturation of oocytes in the presence of brainderived neurotropic factor, one member of neurotropin family, promoted the preimplantational development of bovine embryos (Martins da Silva et al. 2005), the presence of NGF was not detected at any stage in the ovine and bovine preimplantation embryo or within oviduct cells by the reverse transcription polymerase chain reaction (Waston et al. 1992, 1994).It is, however, possible that the technique could not detect a variant mRNA of NGF present at some stages of development. It may be that NGF is used by embryos as a source of protein or nutritional support, with a resultant effect on the development. Our future efforts will be directed to understanding the mechanism of the effect that NGF has on embryonic development in rabbits. References Barboni, B., Mattioli, M., Gioia, L., Turriani, M., Capacchietti, G., Berardinelli, P., Bernabò, N., 2002. Preovulatory rise of NGF in ovine follicular fluid: possible involvement in the control of oocyte maturation. Microscopy Research Technique, 59,516–521 Carney, E.W. and Foote, R.H. 1991. Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, protein-free culture medium. Journal of Reproduction and Fertility, 91, 113–123 Chrenek, P., Makarevich, A.,Vašíček, D., Laurinčík, J., Bulla, J., Gajarska, T. and Rafay, J., 1998. Effects of superovulation, culture and microinjection on development of rabbit embryos in vitro. Theriogenology, 50, 659–666 Corcoran, D., Fair, T. and Lonergan, P., 2005. Predicting embryo quality: mRNA expression and the preimplantation embryo. Reproductive Biomedicine Online, 11, 340–348 Martins da Silva, S.J., Gardner, J.O., Taylor, J.E., Springbett, A., De Sousa, P.A. and Anderson, R.A., 2005. Brain-derived neurotrophic factor promotes bovine oocyte cytoplasmic competence for embryo development. Reproduction, 129,423–434 Desai, N., Lawson, J., and Goldfarb, J., 2000. Assessment of growth factor effects on post-thaw development of cryopreserved mouse morulae to the blastocyst stage. Human Reproduction, 15,410–418 Edwards, R.G., 1995. Physiological and molecular aspects of human implantation.Human Reproduction ,10 (suppl.2), 1–13 Galway, A.B., Oikawa, M., Ny, T. and Hsueh, A.J., 1989. Epidermal growth factor stimulates tissue plasminogen activator activity and messenger ribonucleic acid levels in cultured rat granulosa cells: mediation by pathways independent of protein kinases-A and –C. Endocrinology , 125, 126–135 Giles, J.R. and Foote, R.H., 1997. Effects of gas atmosphere, platelet-derived growth factor and leukemia inhibitory factor on cell numbers of rabbit embryos cultured in a protein-free medium. Reproduction Nutrition Development, 37, 97–104
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