The effects of group size on development and interferon-{tau} secretion by in-vitro fertilized and cultured bovine blastocysts

Melissa A. Larson and H. Michael Kubisch1

Department of Animal Sciences, University of Missouri, Columbia, MO 65211, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effects of culturing bovine embryos in groups were investigated. In the first experiment, 1000 oocytes were matured, fertilized and then cultured in groups of 40 in 25 µl of medium. From half of these groups, blastocysts were removed and cultured separately, while in the other half blastocysts were allowed to remain in the group culture microdrop. Blastocysts developed equally well in both groups, although hatching was reduced in those blastocysts removed from the culture droplet. In the second experiment, 1000 zygotes were cultured from the 8-cell stage to the blastocyst stage either individually or in groups of 40. Culture in groups increased the formation of blastocysts, the percentage of hatching blastocysts, the number of cells within blastocysts and the production of interferon-{tau}. In the final experiment, 1000 zygotes were cultured in groups up to the blastocyst stage. Two-thirds of these blastocysts were then cultured in groups of three, while the remaining blastocysts were cultured individually. Co-culture did not affect hatching or cell number but significantly elevated interferon-{tau} secretion. These results demonstrate that group culture either before or after blastocyst formation can alter the expression of a specific gene important for the establishment of pregnancy.

Key words: bovine blastocyst/culture/embryo density/group size/interferon-{tau}


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preimplantation embryos of many species secrete factors that are required for triggering maternal recognition of pregnancy. In humans, human chorionic gonadotrophin (HCG) production by the trophoblast of the developing conceptus is necessary to maintain function of the corpus luteum. Similarly, secretion of the pregnancy-specific ß-1 glycoprotein (SP1) by the human embryo is thought to prevent rejection of the fetus by the maternal immune system (Bischof, 1984Go). Both HCG and SP1 can be detected in the medium of cultured embryos following in-vitro fertilization (Fishel et al., 1984Go; Dokras et al., 1991Go; Lopata and Oliva, 1993Go; Woodward et al., 1993Go; Saith et al., 1996Go).

In ruminant species, maternal recognition of pregnancy depends on the embryonic secretion of interferon-{tau}. 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., 1989Go; Ott et al., 1993Go). Interferon-{tau} is produced at maximal levels just prior to the time of implantation (Bartol et al., 1985Go; Roberts et al., 1989Go), but can readily be detected in the medium of cultured blastocysts (Hernandez-Ledezma et al., 1992aGo, 1993Go). The properties of interferon-{tau} are similar to those of other interferons: it acts as an antiviral agent with the same host range and potency as interferon-{alpha}; it effectively inhibits proliferation of certain cell types in culture; and it competes with human interferon-{alpha} for binding to the Type I IFN receptor (Stewart et al., 1987Go; Pontzer et al., 1988Go; Hansen et al., 1989Go; Knickerbocker and Niswender, 1989Go; Li and Roberts, 1994Go). However, interferon-{tau} 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., 1990Go; 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., 1993Go; Turner and Lenton, 1996Go), although hatching is not a prerequisite for the production of HCG (Woodward et al., 1993Go, 1994Go). Similarly, interferon-{tau} secretion increases during hatching of bovine blastocysts, a process that is independent of the concomitant increase in cell number (Kubisch et al., 1998Go). Early studies also suggested a correlation between the morphological quality of hatched blastocysts and production of interferon-{tau} (Hernandez-Ledezma et al., 1992aGo, 1993Go), while a more recent study has demonstrated a significant effect of the age at blastocyst formation on the production of interferon-{tau} (Kubisch et al., 1998Go).

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-{tau}, a gene product believed to be important for the establishment of pregnancy.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In-vitro maturation and fertilization
A total of 3000 bovine oocytes were used from eight separate ovary collections (batches). The ovaries were collected at an abattoir and transported to the laboratory at 39°C in phosphate-buffered saline (PBS: 1.4 mmol/l KH2PO4, 8 mmol/l Na2HPO4, 0.14 mol/l NaCl, 1.7 mmol/l KCl) containing 10 mg/l of gentamycin (Sigma, St Louis, MO, USA). Follicles 2–8 mm in size were aspirated, and the follicular fluid was pipetted through a 100 µm cell strainer (Becton Dickinson Labware, Franklin Lakes, NY, USA). Cumulus–oocyte complexes were washed with TALP-HEPES (Parrish et al., 1986Go). Oocytes were matured in TCM199 (Sigma) containing 10% fetal calf serum (Gibco, Grand Island, NY, USA) supplemented with 2 µg/ml of follicle stimulating hormone (FSH), 2 µg/ml oestradiol-17ß and 0.25 mmol/l sodium pyruvate (Sigma; Saeki et al., 1990Go). After 24 h of maturation, oocytes were fertilized as described previously (Hernandez-Ledezma et al., 1992bGo) with frozen–thawed semen that had been subjected to centrifugation (300 g for 30 min at 25°C) through a discontinuous gradient of Percoll (Aitken and Clarkson, 1988Go). Gametes were co-cultured for 18 h.

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., 1972Go). 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-{tau} 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-{tau} concentration.

In Experiment 3, the effects of culturing blastocysts in groups on hatching, cell number and interferon-{tau} 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 10–20 min and fixed with a solution of ethanol:acetic acid:water (3:2:1) at 4°C for 1 min (Fukui and Ono, 1989Go). 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-{tau} 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., 1993Go). The extent of cell protection was compared to that provided by serial dilutions of recombinant bovine interferon-{tau}, which had a specific activity of 5.4x107 IU/mg. All interferon-{tau} values throughout this article are expressed as averages, i.e. all are on a `per embryo' basis.

Statistical analyses
Developmental data were analysed by {chi}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-{tau} 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 Burr–Foster Q-test (Anderson and McLean, 1974Go), interferon values were analysed after log transformation but are reported as raw means with their individual standard errors.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In experiment 1, 908 zygotes cleaved with 782 having reached the 8-cell stage after 48 h in culture. The ovary batch had no effect on the percentage of oocytes cleaving or on the percentage of blastocysts (30.3 versus 29.3%). An equivalent percentage of embryos developed to the blastocyst stage regardless of whether blastocysts remained in their respective culture group (28.4%) or were removed from it (31.0%, Table IGo). Likewise, treatment did not affect the age at which blastocysts formed (8.4 ± 0.1 versus 8.3 ± 0.1 days). However, a significantly larger proportion of blastocysts remaining in their respective culture droplets subsequently hatched in comparison with those that had been removed (44.4 versus 31.0%, P < 0.05).


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Table I. Effect of removal of blastocysts from the medium on the appearance of subsequent blastocysts
 
In experiment 2, a total of 920 oocytes cleaved with 765 having reached the 8-cell stage after 48 h of culture. Whereas the cleavage rate and number of 8-cell embryos were not affected, there was a significant effect of ovary batch on the percentage of oocytes proceeding to the blastocyst stage (30.7 versus 20.8 and 21.3%, P < 0.05). Of the 8-cell embryos, 459 were assigned to individual culture, while the remaining 306 embryos were cultured in groups. Of the group-cultured embryos, 47.4% (145) developed into blastocysts, while significantly fewer (20.5%, P < 0.05, Table IIGo) did so in individual culture. Culture in groups also significantly increased the number of blastocysts that hatched (26.9 versus 12.8%), cell number (78.0 ± 3.6 versus 65.4 ± 4.7) and the mean concentration of interferon-{tau} in the medium droplets (Table IIGo). However, the culture method had no effect on the mean age at blastocyst formation (8.4 ± 0.1 versus 8.2 ± 0.1 days, respectively). The ovary batch also had a significant effect (P < 0.05) on interferon-{tau} production with values of 1392.4 ± 200.5, 1518.1 ± 246.2 and 1364.2 ± 219.7 pmol/l, respectively. Similarly, interferon-{tau} production was significantly affected by the age at blastocyst formation. Droplets from blastocysts resulting from group culture contained 1000.4 ± 279.3, 1275.4 ± 180.7, 1704.2 ± 379.5, 3691.2 ± 757.9 and 2487.2 ± 564.2 pmol/l, those from individual culture 432.1 ± 98.6, 639.3 ± 154.4, 1971.8 ± 727.5, and 1715.6 ± 402.8 and 472.5 pmol/l for days 7 to day 11, respectively (P < 0.01). Although the age at blastocyst formation was not associated with cell number in blastocysts following culture in groups (P = 0.4), individually cultured blastocysts forming late had significantly fewer cells than those appearing early in culture (P < 0.05; Figure 1Go).


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Table II. Effects of culturing 8-cell embryos in groups on interferon-{tau} secretion of individually cultured blastocysts
 


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Figure 1. Least square means of cell numbers of individually cultured embryos. a,bDifferent superscripts indicate cell numbers differ by P < 0.05.

 
Of the 1000 oocytes in the third experiment, 818 cleaved, 721 reached the 8-cell stage and 317 subsequently developed to the blastocyst stage. Ovary batch had no effect on the percentage of oocytes that cleaved or reached the blastocyst stage. Of the 317 blastocysts, 231 were assigned to culture in groups of three, whereas the remaining 86 blastocysts were cultured individually for 48 h. Hatching (38.0 versus 32.6%) and cell number (85.5 ± 5.5 versus 75.6 ± 7.7) were not affected by culture treatment. In contrast, age at blastocyst formation again had a significant effect on interferon-{tau} production. Droplets of group-cultured blastocysts contained 997.5 ± 151.8, 3472.9 ± 538.8, 8150.8 ± 1859.4, 7345.2 ± 3231.4 and 5442.0 ± 2597.2 pmol/l, whereas those from individually cultured blastocysts contained 527.6 ± 164.7, 760.5 ± 143.3, 1699.2 ± 298.8, 1453.2 ± 551.6 and 1012.2 ± 190.2 pmol/l for blastocysts appearing from day 7 to day 11 respectively (P < 0.01). When adjusted for the number of blastocysts cultured within each droplet, blastocysts cultured in groups also secreted significantly more interferon-{tau} into the medium than those cultured individually (1652.6 ± 251.1 versus 1100.0 ± 130.6 pmol/l, P < 0.05, Table IIIGo).


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Table III. Effects of culturing blastocysts in groups or individually on interferon-{tau} secretion
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Overall development in this study, as assessed by the percentage of oocytes reaching the blastocyst stage, is well within the range generally reported for bovine in-vitro matured, fertilized and cultured embryos when SOF medium was used (Keskintepe et al., 1995Go). It is not clear whether the number of bovine blastocysts that can be generated in vitro represents a real limit that may in part arise from a heterogeneous oocyte pool, or whether this limit is a result of sub-optimal maturation or culture systems (Brackett and Zuelke, 1993Go; Blondin and Sirard, 1995Go).

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., 1988Go). 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., 1995Go) 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., 1997Go), ovine (Gardner et al., 1994Go) and murine embryos (Paria and Dey, 1990Go; Lane and Gardner, 1992Go; Kato and Tsunoda, 1994Go; Salahuddin et al., 1995Go). Culturing human embryos in groups is, likewise, thought to enhance their development (Jones et al., 1998Go). It has been suggested (Walters et al., 1985Go) 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., 1995Go) 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, 1990Go) on the development of individually cultured mouse embryos with the addition of epidermal growth factor (EGF) or transforming growth factor (TGF) {alpha} or ß1 to the medium. Indeed, TGF-{alpha} 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, 1991Go; Brison and Schultz, 1997Go). The finding of transcripts for TGF {alpha} and ß2, PDGF A chain and insulin-like growth factor II (IGF-II) in bovine embryos has been reported (Watson et al., 1992Go), as well as identifying receptors for IGF-I, IGF-II and PDGF ({alpha} subunit). In addition, Beauchamp and Croy have demonstrated the presence of colony-stimulating factor-1 receptors on bovine blastocysts (Beauchamp and Croy, 1991Go). 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., 1992Go; Thibodeaux et al., 1993Go; de Moraes and Hansen, 1997Go).

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-{tau} 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-{tau} (Han et al., 1997Go).

It is not known whether human blastocysts produce or secrete interferons, although there are reports that human placental outgrowths produce interferon-{gamma} in vitro (Tao and Cao, 1993Go). Moreover, Whaley et al. (1994) have shown the presence of mRNA in the human placenta whose sequence has some similarity to interferon-{tau}. One secretory product of human blastocysts that has been identified is HCG (Fishel et al., 1984Go). Unlike interferon-{tau}, the secretion of HCG is not affected by the age at which blastocyst formation occurs (Dokras et al., 1991Go), 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., 1998Go).

It is as yet unknown whether variation in interferon-{tau} 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
 
The authors would like to thank Select Sires, Plains City, OH, USA, for donation of the bovine semen. This research was supported by a grant from the National Institutes of Health (HD R29 36421).


    Notes
 
1 To whom correspondence should be addressed at: Transgenic Animal Core Facility, N125 Animal Sciences Research Center, University of Missouri, Columbia, MO 65211, USA Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Anderson, V.L. and McLean, R.A. (1974) Design of Experiments: A Realistic Approach. Marcel Dekker, New York, pp. 22–23.

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, 367–376.[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, 681–894.[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, 811–817.[Abstract]

Bischof, P. (1984) Placental proteins. Contrib. Obstet. Gynecol., 12, 6–22.

Blondin, P. and Sirard, M.-A. (1995) Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes. Mol. Reprod. Dev., 41, 54–62.[ISI][Medline]

Brackett, B.G. and Zuelke, K.A. (1993) Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology, 39, 43–64.[ISI]

Brison, D.R. and Schultz, R.M. (1997) Apoptosis during mouse blastocyst formation: evidence for a role for survival factors including TGF-{alpha}. Biol. Reprod., 56, 1088–1096.[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, 3817–3821.[Abstract]

Dardik, A. and Schultz, R.M. (1991) Blastocoel expansion in the preimplantation mouse embryo: stimulatory effect of TGF-{alpha} and EGF. Development, 113, 919–930.[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, 1060–1065.[Abstract]

Dokras, A., Sargent, I.L., Ross, C. et al. (1991) The human blastocyst: morphology and human gonadotrophin secretion in vitro. Hum. Reprod., 6, 1143–151.[Abstract]

Dokras, A., Sargent, I.L. and Barlow, D.H. (1993) Human blastocyst grading: an indicator of developmental potential? Hum. Reprod., 8, 2119–2127.[Abstract]

Farin, C.E., Imakawa, K., Hansen, T.R. et al. (1990) Expression of trophoblastic interferon genes in sheep and cattle. Biol. Reprod., 43, 210–218.[Abstract]

Fishel, S.B., Edwards, R.G. and Evans, C.J. (1984) Human chorionic gonadotropin secreted by preimplantation embryos cultured in vitro. Science, 223, 816–818.[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, 501–506.[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, 390–400.[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, 4757–4767.[Abstract/Free Full Text]

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, 215–225.[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, 374–380.[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, 1267–1277.[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, 1–6.[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, 169–177.[Abstract]

Kato, Y. and Tsunoda, Y. (1994) Effects of culture density of mouse zygotes on the development in vitro and in vivo. Theriogenology, 41, 1315–1322.[ISI]

Keskintepe, L., Burnley, C.A. and Brackett, B.G. (1995) Production of viable bovine blastocysts in defined in vitro conditions. Biol. Reprod., 52, 1410–1417.[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, 361–369.[Abstract]

Kubisch, H.M., Larson, M.A. and Roberts, R.M. (1998) Relationship between age of blastocyst formation and interferon-{tau} secretion by in-vitro derived bovine embryos. Mol. Reprod. Dev., 49, 254–260.[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, 558–562.[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, 821–826.[Abstract/Free Full Text]

Li, J. and Roberts, R.M. (1994) Interferon-{tau} and interferon-{alpha} interact with the same receptors in bovine endometrium. J. Biol. Chem., 269, 13544–13555.[Abstract/Free Full Text]

Lopata, A. and Oliva, K. (1993) Chorionic gonadotropin secreted by human blastocysts. Hum. Reprod., 8, 932–938.[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, 1645–1652.[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, 161–169.[ISI]

Ott, T.L., van Heeke, G., Hostetler, C.E. et al. (1993) Intrauterine injections of recombinant ovine interferon-{tau} extends the interestrous interval in sheep. Theriogenology, 40, 757–769.[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, 4756–4760.[Abstract]

Parrish, J.J., Susko-Parrish, J.L., Leibfried-Rutledge, I. et al. (1986) Bovine in vitro fertilization with frozen–thawed semen. Theriogenology, 25, 591–601.[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, 801–807.[ISI][Medline]

Roberts, R.M., Imakawa, K., Niwano, Y. et al. (1989) Interferon production by the preimplantation sheep embryo. J. Interferon Res., 9, 175–187.[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, 1035–1039.[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, 1038–1042.[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, 2382–2385.[Abstract]

SAS Institute Inc. (1989) JMP User's Guide. Version 2. Statistical Analysis System, Cary, NC, pp. 243–357.

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, R13–R15.[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, 12–133.

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, 21–212.

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, 493–497.[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, 61–66.[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, 1966–1974.[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, 557–563.[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, 87–95.[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, 10864–10868.[Abstract/Free Full Text]

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, 1069–1074.[ISI]

Woodward, B.J., Lenton, E.A. and Turner, K. (1993) Human chorionic gonadotrophin: embryonic secretion is a time-dependent phenomenon. Hum. Reprod., 8, 1463–1468.[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, 1909–1914.[Abstract]

Submitted on March 2, 1999; accepted on May 20, 1999.