Department of Animal Sciences, University of Missouri, Columbia, MO 65211, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words:
bovine blastocyst/culture/embryo density/group size/interferon-
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In ruminant species, maternal recognition of pregnancy depends on the embryonic secretion of interferon-. Evidence for such a role comes from studies that have demonstrated its ability to extend the oestrous cycle following intrauterine injections in sheep (Stewart et al., 1989
; Ott et al., 1993
). Interferon-
is produced at maximal levels just prior to the time of implantation (Bartol et al., 1985
; Roberts et al., 1989
), but can readily be detected in the medium of cultured blastocysts (Hernandez-Ledezma et al., 1992a
, 1993
). The properties of interferon-
are similar to those of other interferons: it acts as an antiviral agent with the same host range and potency as interferon-
; it effectively inhibits proliferation of certain cell types in culture; and it competes with human interferon-
for binding to the Type I IFN receptor (Stewart et al., 1987
; Pontzer et al., 1988
; Hansen et al., 1989
; Knickerbocker and Niswender, 1989
; Li and Roberts, 1994
). However, interferon-
differs markedly from other interferons in that it is not inducible by virus and is transiently produced only by the embryonic trophoblast (Farin et al., 1990
; Cross et al., 1991).
Given their presumed roles in the establishment of pregnancy, attempts have been made to link these embryonic secretory products with the morphological quality and developmental competence of preimplantation embryos in vitro. In human blastocysts, there appears to be a correlation between their ability to hatch, and HCG production (Dokras et al., 1993; Turner and Lenton, 1996
), although hatching is not a prerequisite for the production of HCG (Woodward et al., 1993
, 1994
). Similarly, interferon-
secretion increases during hatching of bovine blastocysts, a process that is independent of the concomitant increase in cell number (Kubisch et al., 1998
). Early studies also suggested a correlation between the morphological quality of hatched blastocysts and production of interferon-
(Hernandez-Ledezma et al., 1992a
, 1993
), while a more recent study has demonstrated a significant effect of the age at blastocyst formation on the production of interferon-
(Kubisch et al., 1998
).
As in other embryo culture systems, bovine embryos are generally cultured in groups. Although there are beneficial effects on overall development, it is unclear whether such culture conditions can affect the activity of specific genes. Here we show that culturing bovine embryos in groups not only affects their developmental fate, but also significantly alters the secretion of interferon-, a gene product believed to be important for the establishment of pregnancy.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Embryo culture and experimental design
Following insemination, presumptive zygotes were washed, vortexed to remove cumulus cells and transferred into synthetic oviductal fluid (SOF) supplemented with 10% fetal calf serum and 1 mmol/l glutamine (Tervit et al., 1972). Zygotes were cultured in groups of 40 in 25 µl of medium. After 48 h in culture, embryos were examined and the rate of cleavage was assessed. Eight-cell embryos were returned to fresh droplets of SOF and cultured in groups of 35. Approximately 75% of the medium was subsequently replaced at 48 h intervals. Between days 6 and 11, droplets were examined daily for the appearance of blastocysts.
Experiment 1 was designed to investigate the possible inhibitory effects of blastocysts upon the development of less-advanced embryos in the same culture droplet. A total of 1000 embryos from two batches of ovaries were divided into two treatment groups. In treatment group 1, expanded blastocysts were removed daily, transferred to fresh droplets and cultured in groups of 20. In the second treatment group, blastocysts remained in their respective medium droplets, although these droplets were examined daily and the appearance of expanded blastocysts was noted.
Experiment 2 was performed to assess the effects of individual culture on development and interferon- secretion. One thousand zygotes from three ovary collections were again cultured to the 8-cell stage as described above. At that point, they were separated and randomly assigned to either individual culture in 10 µl or to culture in groups of 35 in 25 µl of medium. Significantly more 8-cell embryos were assigned to individual culture as it was expected that fewer would reach the blastocyst stage. Between days 7 and 11, expanded blastocysts were removed daily from the culture droplet and cultured individually in droplets of 40 µl for 48 h, after which time the number of hatched blastocysts was recorded. Blastocysts were then fixed for cell counts and the culture medium was frozen for later assessment of interferon-
concentration.
In Experiment 3, the effects of culturing blastocysts in groups on hatching, cell number and interferon- secretion were examined. Again, 1000 zygotes from three ovary collections were cultured to the blastocyst stage as described in the experimental design. Expanded blastocysts were again removed daily and subsequently either cultured individually or in groups of three in 40 µl of medium for 48 h. Blastocysts and medium droplets were treated as in the preceding experiment.
It should be noted that the constraints of the three experimental designs prevented maintenance of a constant embryo number to volume of medium ratio. However, we know of no evidence which suggests that this ratio has any effect on the development of individually cultured bovine embryos. A culture volume of 10 µl for individual embryos (experiment 2) was chosen for most efficient utilization of space in culture dishes and to prevent accidental removal of the embryo when the medium was changed. Blastocysts were always cultured in 40 µl droplets because that was the amount of medium our antiviral assays were designed to accept.
Determination of cell number
Embryos were placed into 0.9% (v/v) of sodium citrate (in water) for 1020 min and fixed with a solution of ethanol:acetic acid:water (3:2:1) at 4°C for 1 min (Fukui and Ono, 1989). The fixed embryos were transferred to slides, air-dried for at least 1 h and stained with 10% (w/v) Giemsa (Sigma). Cell numbers were determined on an inverted microscope by using Hoffman optics.
Determination of interferon- concentration in embryo culture medium
Embryo culture medium was assayed for antiviral activity by using a cytopathic reduction assay that relies on the use of a bovine kidney cell line (MDBK cells, ATCC CCL 22) challenged with a vesicular stomatitis virus (Hernandez-Ledezma et al., 1993). The extent of cell protection was compared to that provided by serial dilutions of recombinant bovine interferon-
, which had a specific activity of 5.4x107 IU/mg. All interferon-
values throughout this article are expressed as averages, i.e. all are on a `per embryo' basis.
Statistical analyses
Developmental data were analysed by 2 procedures. Data on cell number and interferon concentrations in the medium samples were analysed by least square analyses, and individual means were compared by orthogonal contrast (JMP software of SAS; SAS Institute, 1989). The statistical model for the analyses of interferon-
concentrations included ovary batch, age at blastocyst formation, treatment, hatching and cell number as a covariable. Because of heterogeneity of variance, as determined by a BurrFoster Q-test (Anderson and McLean, 1974
), interferon values were analysed after log transformation but are reported as raw means with their individual standard errors.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of the first experiment indicate that the presence of blastocysts in a group of embryos does not appear to inhibit the development of less-advanced embryos. It has been proposed that in the pig, faster developing embryos might be able to reduce the survival in utero of those embryos developing more slowly (Wilde et al., 1988). It is unclear whether such a mechanism exists in other species, but our observations do not support an inhibitory effect of advanced embryos on those developing more slowly. It has been shown (Salahuddin et al., 1995
) that the presence of degenerating mouse oocytes in a culture drop significantly reduced the number of embryos developing to the blastocyst stage. The majority of embryos in the present study did not reach the blastocyst stage and were consequently degenerating. Although we did not assess the effect of these embryos on overall development, it is clear that hatching was not affected by their presence. The significant increase in hatching among blastocysts remaining in the culture droplet may be due to the presence of paracrine factors secreted by other embryos in the cohort, which `condition' the medium. Blastocysts removed from the cohort, on the other hand, may not effectively condition the fresh medium droplet to which they were transferred.
The beneficial effects of culturing embryos in groups on blastocyst formation, size and hatching seen in the second experiment supports earlier evidence from bovine (O'Doherty et al., 1997), ovine (Gardner et al., 1994
) and murine embryos (Paria and Dey, 1990
; Lane and Gardner, 1992
; Kato and Tsunoda, 1994
; Salahuddin et al., 1995
). Culturing human embryos in groups is, likewise, thought to enhance their development (Jones et al., 1998
). It has been suggested (Walters et al., 1985
) that there is a positive interaction among human embryos in vivo which accounts for a disproportionate increase in the number of implantations when more than one embryo is transferred. The mitogenic effects of group culture are most likely to be mediated through the secretion of paracrine growth factors that are not present in sufficient concentration in the fetal calf serum. It has been shown (Thibodeaux et al., 1995
) that addition of platelet-derived growth factor (PDGF) can overcome the developmental retardation of individually cultured bovine embryos. Similarly, a beneficial effect has been reported (Paria and Dey, 1990
) on the development of individually cultured mouse embryos with the addition of epidermal growth factor (EGF) or transforming growth factor (TGF)
or ß1 to the medium. Indeed, TGF-
appears to play an important role in stimulating mitotic activity in murine embryos as well as in reducing apoptotic cell death in both the trophectoderm and the inner cell mass (Dardik and Schultz, 1991
; Brison and Schultz, 1997
). The finding of transcripts for TGF
and ß2, PDGF A chain and insulin-like growth factor II (IGF-II) in bovine embryos has been reported (Watson et al., 1992
), as well as identifying receptors for IGF-I, IGF-II and PDGF (
subunit). In addition, Beauchamp and Croy have demonstrated the presence of colony-stimulating factor-1 receptors on bovine blastocysts (Beauchamp and Croy, 1991
). However, the effects of adding growth factors to bovine embryo culture media have been small or negligible, although PDGF and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been shown to enhance development to the blastocyst stage (Larson et al., 1992
; Thibodeaux et al., 1993
; de Moraes and Hansen, 1997
).
It is interesting to note that the beneficial effect of culturing embryos in groups on cell number and hatching ability was observed only when embryos were cultured in groups prior to blastocyst formation. Individual culture of blastocysts did not reduce cell number or hatching when compared to blastocysts that were cultured in groups of three. This suggests that the developmental fate has been established at blastocyst formation, although it is possible that either the blastocyst number within groups or the length of time in culture were insufficient to result in any effects on development. On the other hand, group-cultured blastocysts did secrete significantly more interferon- than those cultured individually. The cause of this increase remains unknown, but it is not likely to be the result of a positive feedback mechanism, because blastomeres do not possess receptors to bind and internalize interferon-
(Han et al., 1997
).
It is not known whether human blastocysts produce or secrete interferons, although there are reports that human placental outgrowths produce interferon- in vitro (Tao and Cao, 1993
). Moreover, Whaley et al. (1994) have shown the presence of mRNA in the human placenta whose sequence has some similarity to interferon-
. One secretory product of human blastocysts that has been identified is HCG (Fishel et al., 1984
). Unlike interferon-
, the secretion of HCG is not affected by the age at which blastocyst formation occurs (Dokras et al., 1991
), but a recent report has demonstrated that the type of substrate on which human blastocysts are cultured during outgrowth formation can affect HCG production (Martin et al., 1998
).
It is as yet unknown whether variation in interferon- production in blastocysts can have long-lasting effects on the establishment and maintenance of pregnancy. However, our results show that the expression of specific genes can be affected by the presence of other embryos during culture in vitro. If the products of such genes are involved in determining the subsequent fate of the embryo, a careful assessment of culture conditions and their effects on gene expression is warranted.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aitken, R.J. and Clarkson, J.S. (1988) Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J. Androl., 9, 367376.[Abstract]
Bartol, F.F., Roberts, R.M., Bazer, F.W. et al. (1985) Characterization of proteins produced by peri-attachment bovine conceptuses. Biol. Reprod., 32, 681894.[Abstract]
Beauchamp, J.L. and Croy, B.A. (1991) Assessment of expression of the receptor for colony-stimulating factor-1 (FMS) in bovine trophoblast. Biol. Reprod., 45, 811817.[Abstract]
Bischof, P. (1984) Placental proteins. Contrib. Obstet. Gynecol., 12, 622.
Blondin, P. and Sirard, M.-A. (1995) Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes. Mol. Reprod. Dev., 41, 5462.[ISI][Medline]
Brackett, B.G. and Zuelke, K.A. (1993) Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology, 39, 4364.[ISI]
Brison, D.R. and Schultz, R.M. (1997) Apoptosis during mouse blastocyst formation: evidence for a role for survival factors including TGF-. Biol. Reprod., 56, 10881096.[Abstract]
Cross, J.C. and Roberts, R.M. (1991) Constitutive and trophoblast-specific expression of a class of bovine interferon genes. Proc. Natl. Acad. Sci. USA, 88, 38173821.[Abstract]
Dardik, A. and Schultz, R.M. (1991) Blastocoel expansion in the preimplantation mouse embryo: stimulatory effect of TGF- and EGF. Development, 113, 919930.[Abstract]
de Moraes, A.A.S. and Hansen, P.J. (1997) Granulocyte-macrophage colony-stimulating factor promotes development of in vitro produced bovine embryos. Biol. Reprod., 57, 10601065.[Abstract]
Dokras, A., Sargent, I.L., Ross, C. et al. (1991) The human blastocyst: morphology and human gonadotrophin secretion in vitro. Hum. Reprod., 6, 1143151.[Abstract]
Dokras, A., Sargent, I.L. and Barlow, D.H. (1993) Human blastocyst grading: an indicator of developmental potential? Hum. Reprod., 8, 21192127.[Abstract]
Farin, C.E., Imakawa, K., Hansen, T.R. et al. (1990) Expression of trophoblastic interferon genes in sheep and cattle. Biol. Reprod., 43, 210218.[Abstract]
Fishel, S.B., Edwards, R.G. and Evans, C.J. (1984) Human chorionic gonadotropin secreted by preimplantation embryos cultured in vitro. Science, 223, 816818.[ISI][Medline]
Fukui, Y. and Ono, H. (1989) Effects of sera, hormones and granulosa cells added to culture medium for in vitro maturation, fertilization, cleavage and development of bovine oocytes. J. Reprod. Fertil., 86, 501506.[Abstract]
Gardner, D.K., Lane, M., Spitzer, A. and Batt, P. (1994) Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: amino acids, vitamins and culturing embryos in groups stimulate development. Biol. Reprod., 50, 390400.[Abstract]
Han, C.-S., Mathialagan, N., Kleman, S.W. and Roberts, R.M. (1997) Molecular cloning of ovine and bovine type I interferon receptor subunits from uteri, and endometrial expression of messenger ribonucleic acid for ovine receptors during the estrous cycle and pregnancy. Endocrinology, 138, 47574767.
Hansen, T.R., Kazemi, M., Keisler, D.H. et al. (1989) Complex binding of the embryonic interferon ovine trophoblast protein-1 to endometrial receptors. J. Interferon Res., 9, 215225.[ISI][Medline]
Hernandez-Ledezma, J.J., Sikes, J.D., Murphy, C.N. et al. (1992a) Expression of bovine trophoblast interferon in conceptuses derived by in vitro techniques. Biol. Reprod., 47, 374380.[Abstract]
Hernandez-Ledezma, J.J., Villanueva, C., Sikes, J.D. and Roberts, R.M. (1992b) Effects of CZB vs medium 199 and of conditioning with either bovine oviductal epithelial cells or buffalo rat liver cells on the development of bovine zygotes derived by in vitro maturation- in vitro fertilization procedures. Theriogenology, 39, 12671277.[ISI]
Hernandez-Ledezma, J.J., Mathialagan, N., Villanueva, C. et al. (1993) Expression of bovine trophoblast interferons by in vitro-derived blastocysts is correlated with their morphological quality and stage of development. Mol. Reprod. Dev., 36, 16.[ISI][Medline]
Jones, G.M., Trounson, A.O., Gardner, D.K. et al. (1998) Evolution of a culture protocol for successful blastocyst development and pregnancy. Hum. Reprod., 13, 169177.[Abstract]
Kato, Y. and Tsunoda, Y. (1994) Effects of culture density of mouse zygotes on the development in vitro and in vivo. Theriogenology, 41, 13151322.[ISI]
Keskintepe, L., Burnley, C.A. and Brackett, B.G. (1995) Production of viable bovine blastocysts in defined in vitro conditions. Biol. Reprod., 52, 14101417.[Abstract]
Knickerbocker, J.J. and Niswender, G.D. (1989) Characterization of endometrial receptors for ovine trophoblast protein-1 during the estrous cycle and early pregnancy in the sheep. Biol. Reprod., 40, 361369.[Abstract]
Kubisch, H.M., Larson, M.A. and Roberts, R.M. (1998) Relationship between age of blastocyst formation and interferon- secretion by in-vitro derived bovine embryos. Mol. Reprod. Dev., 49, 254260.[ISI][Medline]
Lane, M. and Gardner, D.K. (1992) Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro. Hum. Reprod., 7, 558562.[Abstract]
Larson, R.C., Ignotz, G.G. and Currie, W.B. (1992) Platelet derived growth factor (PDGF) stimulates development of bovine embryos during the fourth cell cycle. Development, 115, 821826.
Li, J. and Roberts, R.M. (1994) Interferon- and interferon-
interact with the same receptors in bovine endometrium. J. Biol. Chem., 269, 1354413555.
Lopata, A. and Oliva, K. (1993) Chorionic gonadotropin secreted by human blastocysts. Hum. Reprod., 8, 932938.[ISI][Medline]
Martin K.L., Barlow, D.H. and Sargent, I.L. (1998) Heparin-binding epidermal growth factor significantly improves human blastocyst development and hatching in serum-free medium. Hum. Reprod. 13, 16451652.[Abstract]
O'Doherty, E.M., Wade, M.G., Hill, J.L. and Boland, M.P. (1997) Effects of culturing bovine oocytes either singly or in groups on development to blastocysts. Theriogenology, 48, 161169.[ISI]
Ott, T.L., van Heeke, G., Hostetler, C.E. et al. (1993) Intrauterine injections of recombinant ovine interferon- extends the interestrous interval in sheep. Theriogenology, 40, 757769.[ISI]
Paria, B.C. and Dey, S.K. (1990) Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors. Proc. Natl. Acad. Sci. USA, 87, 47564760.[Abstract]
Parrish, J.J., Susko-Parrish, J.L., Leibfried-Rutledge, I. et al. (1986) Bovine in vitro fertilization with frozenthawed semen. Theriogenology, 25, 591601.[ISI]
Pontzer, C.H., Torres, B.A., Vallet, J.L. and Bazer, F.W. (1988) Antiviral activity of the pregnancy recognition hormone ovine trophoblast protein-1. Biochem. Biophys. Res. Commun., 152, 801807.[ISI][Medline]
Roberts, R.M., Imakawa, K., Niwano, Y. et al. (1989) Interferon production by the preimplantation sheep embryo. J. Interferon Res., 9, 175187.[ISI][Medline]
Saeki, K., Hoshi, M., Leibfried-Rutledge, M.L. and First, N.L. (1990) In vitro fertilization and development of bovine oocytes matured with commercially available follicle stimulation hormone. Theriogenology, 34, 10351039.[ISI]
Saith, R.R., Bersinger, N.A., Barlow, D.H. and Sargent, I.L. (1996) The role of pregnancy-specific ß-1 glycoprotein (SP1) in assessing human blastocyst quality in vitro. Hum. Reprod., 11, 10381042.[Abstract]
Salahuddin, S., Ookutsu, S., Goto, K. et al. (1995) Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in vitro fertilized mouse embryos. Hum. Reprod., 10, 23822385.[Abstract]
SAS Institute Inc. (1989) JMP User's Guide. Version 2. Statistical Analysis System, Cary, NC, pp. 243357.
Stewart, H.J., McCann, S.H., Barker, P.J. et al. (1987) Interferon sequence homology and receptor binding activity of ovine trophoblast antiluteolytic protein. J. Endocrinol., 115, R13R15.[Abstract]
Stewart, H.J., Flint, A.P.F., Lamming, G.E. et al. (1989) Antiluteolytic effects of blastocyst-secreted interferon investigated in vitro and in vivo in the sheep. J. Reprod. Fertil., 37, 12133.
Tao, Y.X. and Cao, Y.Q. (1993) Modulation of interferon secretion by concanavalin A and interleukin-2 in first trimester placental explants in vitro. J. Reprod. Immunol., 24, 21212.
Tervit, H.R., Whittingham, D.G. and Rowson, L.E.R. (1972) Successful culture in vitro of sheep and cattle ova. J. Reprod. Fertil., 30, 493497.[Medline]
Thibodeaux, J.K., del Vecchio, R.P. and Hansel, W. (1993) Role of platelet-derived growth factor in development of in vitro matured and in vitro fertilized bovine embryos. J. Reprod. Fertil., 98, 6166.[Abstract]
Thibodeaux, J.K., Myers, M.W. and Hansel, W. (1995) The beneficial effects of incubating bovine embryos in groups are due to platelet-derived growth factor. Theriogenology, 43, 336 (abstract).
Turner, K. and Lenton, E.A. (1996) The influence of Vero cell culture on human embryo development and chorionic gonadotrophin production in vitro. Hum. Reprod., 11, 19661974.[Abstract]
Walters, D.E., Edwards, R.G. and Meistrich, M.L. (1985) A statistical evaluation of implantation after replacing one or more human embryos. J. Reprod. Fertil., 74, 557563.[Abstract]
Watson, A.J., Hogan, A., Hahnel, A. et al. (1992) Expression of growth factor ligand and receptor genes in the preimplantation bovine embryo. Mol. Reprod. Dev., 31, 8795.[ISI][Medline]
Whaley, A.E., Meka, C.S., Harbison, L.A. et al. (1994) Identification and cellular localization of unique interferon mRNA from human placenta. J. Biol. Chem., 269, 1086410868.
Wilde, M.H., Xie, S., Day, M.L. and Pope, W.F. (1988) Survival of small and large littermate blastocysts in swine after synchronous and asynchronous transfer procedures. Theriogenology, 30, 10691074.[ISI]
Woodward, B.J., Lenton, E.A. and Turner, K. (1993) Human chorionic gonadotrophin: embryonic secretion is a time-dependent phenomenon. Hum. Reprod., 8, 14631468.[Abstract]
Woodward, B.J., Lenton, E.A., Turner, K. and Grace, W.F. (1994) Embryonic human chorionic gonadotrophin secretion and hatching: poor correlation with cleavage rate and morphological assessment during preimplantation development in vitro. Hum. Reprod., 9, 19091914.[Abstract]
Submitted on March 2, 1999; accepted on May 20, 1999.