Development of preimplantation goat (Capra hircus) embryos in vivo and in vitro

Transcription

1 Development of preimplantation goat (Capra hircus) embryos in vivo and in vitro D. Sakkas, P. A. Batt and A. W. N. Cameron Centre for Early Human Development, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia Summary. Preimplantation goat embryos were cultured with or without goat oviduct epithelial cells in Earle's 199 medium + 10% goat serum (E %GS), and in three different simple chemically defined media. In-vivo development was characterized by an extended 8- to 16-cell stage followed by a rapid cleavage rate in the next 3 cell cycles. Culture of 1\p=n-\8-cellembryos in Medium E %GS led to cleavage arrest at the 8\p=n-\16-cellstage, while in the chemically defined media embryos developed poorly and a high percentage failed to pass the 8\p=n-\16-cellstage. In co-culture, however, a high percentage (77% and 96%) of 1\p=n-\2-celland 4\p=n-\8-cellembryos, respectively, developed beyond the 16-cell stage. In co-culture, 1\p=n-\2-cellembryos maintained cleavage rates equivalent to those in vivo until the 8-cell stage, but thereafter cell numbers lagged behind those in vivo, and by 168 h after ovulation, cell numbers (\m=+-\s.e.)in vitro were 47\m=.\6\m=+-\7\m=.\9compared to 238 \m=+-\27\m=.\2in vivo (t 6\m=.\93,P = < 0\m=.\001).The results demonstrate that co-culture of embryos with oviduct cells allows a high percentage of embryos to develop through the period of cleavage arrest, providing a favourable environment for development through the 1\p=n-\16-cellstages but a less adequate environment for development to the blastocyst stage. Keywords: goat; embryo; cleavage rates; oviduct cells; cleavage arrest Introduction The culture of early stage mammalian embryos for prolonged periods in vitro is characterized by a general retardation in development compared to that in vivo (Tervit & Rowson, 1974; Harlow & Quinn, 1982; Cummins et al, 1986). The development in culture of embryos from farm animals from the morula to the blastocyst stages occurs readily in a variety of different media (reviewed by Wright & Bondioli, 1981) but earlier stage embryos frequently block during culture at a stage specific to the species (Bavister, 1988). Co-culture of embryos with oviduct cells has enabled a large proportion of sheep and cattle embryos to develop through these specific cell cycles in which development did not occur in chemically defined media (Eyestone et al, 1987; Gandolfi & Moor, 1987). In the goat there are no detailed reports on the cleavage rates of preimplantation embryos in vivo or in vitro. The limited data available suggest that Day 5-7 goat morulae and early blastocysts (Day 1 = day of mating) can be cultured in Dulbecco's phosphate buffer with 25% goat serum (Bilton & Moore, 1976), whereas Wright & Bondioli (1981) reported that to 8-cell goat embryos failed to develop past the morula stage when cultured in Whitten's medium or Hams FIO supplemented with 1-5% bovine serum albumin or 10% fetal calf serum. This indicates that goat embryos may have an in-vitro block at the early morula stage, in which an oviduct co-culture system may facilitate development as in sheep and cattle (Eyestone et al, 1987; Gandolfi & Moor, 1987).

2 In the present study we present a detailed description of the cell number and rates of development of goat embryos in vivo. The development of early stage goat embryos in simple chemically defined media and in a goat oviduct epithelial cell co-culture system was also studied and the development of preimplantation goat embryos through various cleavage and developmental stages in vitro is reported. Materials and Methods Superovulation and collection ofembryos. Embryos were collected from Angora-Cashmere cross goats run under field conditions at Rye, Victoria, Australia, between March and July Ovulation was synchronized by inserting intravaginal progestagen-impregnated sponges (Repromap, Upjohn Pty Ltd, Sydney, Australia) containing 60 mg medroxyprogesterone acetate for 16 days and then administering 50 pg gonadotrophin-releasing hormone (GnRH, Intervet, Lyppard, Victoria, Australia) intramuscularly 20 h after sponge withdrawal. Ovulation in the goat occurs 24 h after GnRH injection (Cameron et al, 1988). Multiple ovulation was achieved by administering folliclestimulating hormone (FSH-P, Burns Biotech., Heriot Agvet, Victoria, Australia) six times, 12 h apart starting 48 h before sponge withdrawal, with consecutive injections containing 4, 3, 3, 3, 2 and 1 mg FSH. Goats were hand mated at 12-h intervals from GnRH injection until cessation of standing oestrus. Embryos were collected via mid-ventral laparotomy in a flushing medium of Dulbecco's phosphate buffer supplemented with 10% goat serum (Db + 10%GS). The embryos were rinsed in fresh Medium Db + 10%GS and transported at 30 C in Medium Db + 10%GS before transfer to the appropriate culture medium. Preparation of oviduct epithelial cultures. Oviducal cells were obtained from the oviduct flushings of goats 1-3 days after ovulation. Cells were collected in 10-ml centrifuge tubes and transported in Medium Db + 10%GS. Cultures were prepared ~l-5h after collection. The flushings were centrifuged at 600g and the cells resuspended in Earles 199 medium (Flow Laboratories, Sydney, Australia) supplemented with 10% goat serum (E %GS). The oviduct cell suspension was drawn through a 21-gauge needle to dissociate the cell clumps, centrifuged as before, and then resuspended in 4 ml Medium E %GS. Cells were then placed in 0-5-ml samples in Linbro 24-well plates (Flow Laboratories) so that they would reach 70-80% confluence overnight. The following morning the medium was withdrawn from the cell monolayer and centrifuged at 600 g for 5 min to remove unattached cells. The monolayer was washed twice with Medium E %GS and the same 0-5 ml medium was replaced over the cells after centrifugation. All cultures were maintained at 37 C in an atmosphere of 5% C02 in air. In preliminary exper iments, oviduct flushings were centrifuged and dissociated as above and placed on a Ficol density gradient. By taking 0-5 ml samples at the various densities, 85-90% of the cells were found in the higher (10-20%) Ficol densities where epithelial cells are expected to layer (Salamonsen et al, 1985). As the oviduct flushings were found to contain predominantly epithelial cells, a Ficol preparation was not utilized to prepare cultures in the studies reported in this paper. Experiment I. Embryos were collected from superovulated goats 8, 16, 24, 36, 48, 60, 72, 84, 96, 120, 144 and 168 h after ovulation. Cleavage-stage embryos were all placed in culture after noting their developmental stage, while embryos with 8 or more cells were either assessed for cell number or placed in culture to assess their ability to develop in vitro. Embryos at the 1 8-cell stage were cultured for 72 h in Quinn's Human Tubai Fluid (HTF: Quinn et al, 1985), bicarbonate-buffered medium (BM: Moore, 1982), a medium similar to HTF but lacking pyruvate, lactate and glucose, or Medium T6 (Quinn et al, 1982). Embryos with more than 8-16 cells collected at 84 and 96 h after ovulation were cultured for 72 h in Medium BM before assessment for cell number. All media were supplemented with 10% pooled goat serum. A single batch of goat serum was prepared for all experiments by pooling jugular vein serum of several conscious mature does. The serum was heat-inactivated at 56 C for 30 min and stored at 20QC. Embryos were cultured in plastic Petri dishes (Bunzl, Labco Pty Ltd, Melbourne, Australia) in 0-5 ml medium under lightweight paraffin oil (BDH) at 37 C in an atmosphere of 5% C02 in air. Experiment 2. Embryos were collected at h after ovulation to obtain I-2-cell embryos, h after ovulation to obtain 4-8-cell embryos, and 96 h after ovulation to obtain morulae with cells. Embryos were removed from Medium Db + 10%GS and randomly allocated to Medium E %GS or co-culture. Medium E %GS was equilibrated in Linbro wells at 37 C and 5% C02 under oil overnight, before embryo culture. Co-cultured goat embryos were transferred to freshly prepared wells containing oviduct epithelial cells every 36^48 h to obtain the maximum benefit from the oviduct cells and to limit their exposure to alterations in ph caused by metabolism of the cells after prolonged culture. Assessment ofembryo development. All embryos were assessed for stage of development by bright-field microscopy ( 40). Cell numbers were assessed by bright-field microscopy for cleavage-stage embryos, and also by the cell spreading method of Kola et al (1986) for cleavage-stage and compacted embryos. The cell spreading technique involved exposing embryos to a hypotonie solution (0-5% tri-sodium citrate) and transferring them to a clean glass slide. Methanol/glacial acetic acid fixative (3:1, v/v) was dropped onto the embryo while it was blown flat and air dried. Cell nuclei were then stained with 10% Giemsa (BDH Chemicals, Melbourne, Australia) in phosphate buffer for 15 min.

3 Before compaction estimates of cell number were made every 24 h by bright-field microscopy. Embryos in co-culture began to compact at the 8-cell stage, and so cell numbers were assessed by removing 4-8 embryos every 24 h, while in the culture medium alone embryos were removed at 120 h and 144 h after ovulation and assessed for cell number. The viability of 16 I 2-cell embryos cultured in Medium E %GS and cell embryos in co-culture with oviduct cells was assessed at 24-h intervals, beginning at 120 h after ovulation, just before assessment for cell number, by exposure to fluorescein diacetate (FDA). The FDA technique was adapted from that used by Mohr & Trounson (1980). Briefly, embryos were exposed to a solution of 2-5 pg FDA/ml Medium M2 (Quinn et al, 1982) for 1 min, washed in Medium M2 and individually examined under a Nikon fluorescent microscope. Embryos that displayed no fluorescence were regarded as non-viable while embryos that had pale areas were considered to contain some non-viable cells. Statistical analyses. Statistical analyses on the comparison of mean cell number of embryos developing in different treatments were performed using Student's t test. Results Experiment 1 Development ofgoat embryos in vivo. As shown in Fig. 1 the first cleavage division did not occur until h after ovulation, but the following two divisions occurred at about 12-h intervals. At 24 h after ovulation 7/26 (27%) embryos collected had cleaved to 2-cells while by 60 h after ovula tion 23/26 (88%) embryos collected had 8-cells. The first 16-cell embryo was collected 84 h after ovulation (1/14; 7%) and by 96 h after ovulation 12/20 (60%) of the embryos collected had 16-cells or more. Thus, the 8- to 16-cell stage was prolonged compared to the previous two cell cycles and lasted about h. Embryos also began to compact at the 8 16-cell stage. The following 3 cell cycles occurred more rapidly lasting between 10 and 12 h. Early blastocysts were first collected 120 h after ovulation while at 168 h after ovulation all embryos collected were hatched blastocysts. Development of embryos in Media HTF, BM and T6. Development in these media was poor. Only 6/57 (10%), 2/32 (6%) and 0/18 (0%) of the 1-8-cell stage embryos developed past the 8-16-cell stage in Media BM, HTF and T6, respectively. Of the 8-16-cell stage embryos collected at 84 h after ovulation and cultured in Medium BM, 5/16 (31 %), developed past the 16-cell stage with a mean cell number of while 18/21 (85%) embryos collected at 96 h after ovulation developed to the blastocyst stage and had a mean cell number of 103 ± 24-2 per embryo after 72 h in culture (Fig. lc). Experiment 2 Embryos cultured in Medium E %GS. Of the 25 embryos cultured from the 1 2-cell stage in Medium E %GS alone, none developed to the 16-cell stage (Fig. la). Most embryos ceased cleavage at the 8-cell stage (17/25; 68%), and only 2/25 (8%), developed beyond the 8-cell stage. Even so, of the 16 embryos cultured in medium alone that were treated with FDA 14/16 (88%) fluoresced evenly after 120 h in culture, while the remaining 2 were pale in areas. This indicates that the embryos were still metabolically active and capable of cleaving fluorescein from diacetate, i.e. the cellular esterase enzymes remained intact and the cells alive (Mohr & Trounson, 1980). When 4-8-cell embryos were cultured in medium alone, 15/16 (94%) failed to complete the next cleavage stage and the remaining embryo cleaved to the 16-cell stage after 72 h in culture (Fig. lb). Co-culture of 1-2-cell stage goat embryos. After 120 h in co-culture, 36/47 (77%) of the 1-2-cell stage embryos had developed to the 16-cell stage or beyond (Fig. la), and of the 19 embryos examined for their viability by exposure to FDA, at the conclusion of culture, all fluoresced.

4 ), Time (h) after ovulation Fig. 1. The log2 mean cell number (±s.e.) of preimplantation goat embryos developing in vivo ( ) as compared to in-vitro development from (a) the 1 2-cell stage, (b) the 4-8-cell stage and (c) the morula stage, in co-culture with oviduct cells (-- --), Medium E %GS ( -D- and from the morula stage in bicarbonate medium (BM) (-- --). When compared to development observed in vivo, goat embryos in co-culture maintained a normal cleavage rate to the 8-cell stage (Fig. la). At 72 h after ovulation there was no significant difference between mean cell number of co-cultured 1 2-cell stage embryos and embryos of similar age grown in vivo. In contrast, 1 2-cell embryos cultured in medium alone had significantly fewer cells at this stage (t 701, = < 0001). The time to cleave from the 8- to 16-cell stage in co-culture was approximately h which was about 30 h longer than the time taken in vivo. Embryo development in co-culture became progressively more retarded after the 8-cell stage so that after 144 h of culture the cell numbers of 1 2-cell embryos developing in co-culture (47-6 ± 7-9) were approximately one-fifth of those in vivo (238-3 ± 27-2; t 6-93, < 0001) (Fig. la). This = retardation in development was also evident when comparing the morphology of embryos. At 168 h after ovulation, blastocysts had already hatched from their zona pellucida in vivo while at the comparable stage in co-culture 12/22 (55%) remained as compacted morulae and only one had hatched from the zona (Table 1).

5 - Table 1. Morphological stage of development of goat embryos in vivo and after culture at 168 h after ovulation No. of embryos and stage of development Culture method Stage of development at start of culture Total number of embryos Early blastocyst Expanded blastocyst Hatched blastocyst Co-culture Co-culture Co-culture Medium EI % GS Medium BM Grown in vivo 1-2 cells 4-8 cells Co-culture of 4-8-cell stage embryos. After 48 h co-culture, 18/19 (95%) 4-8-cell stage embryos developed beyond the 16-cell stage but they failed to show the rapid cleavage observed for embryos developing in vivo between 84 and 120 h after ovulation (Fig. lb). There was no difference in the rate of development of embryos cultured from the 1 to 2-cell stage or 4- to 8-cell stage in co-culture. Embryos cultured from both stages did not differ significantly in cell numbers or morphology (Table 1) at any given time after ovulation. Culture ofpost-compaction embryos. Compacted embryos cultured for 72 h in medium alone or in co-culture with oviduct cells showed no significant differences in their morphological appearance after culture (Table 1). There was, however, a significant increase (t 2-38, = < 005) in the mean number of cells (±s.e.) of embryos co-cultured with oviduct cells ( ) when compared to those cultured in medium alone ( ), but not when compared to embryos developing from the same cell stage in Medium BM. Cell numbers of embryos in vivo ( ) were significantly greater than those developing from the morula stage in co-culture (82-5 ± 170; t 4-87, = < 0001) and Medium BM ( ; t 3-97, = < 0001) at 168 h after ovulation (Fig. lc). Discussion Cleavage of goat embryos in vivo is characterized by an extended 8-16-cell stage followed by a period of rapid cleavage after compaction. A similar prolonged 8-16-cell stage has been demon strated in the sheep, and is associated with activation of the embryonic genome (Crosby et al, 1988). The lengthened cleavage stage in vivo and its link to embryonic genome activation is also evident in the mouse at the 2-cell stage (Goddard & Pratt, 1983) and the pig at the 4-cell stage (reviewed by Davis, 1985). In a number of species, the stage of embryonic development that coincides with activation of the embryonic genome has also been susceptible to blocks in culture. For example, sheep embryos are difficult to culture through the 8-16-cell stage, certain strains of mice block at the 2-cell stage and pig embryos block at the 4-cell stage in vitro (reviewed by Bavister, 1988). In our study it appeared that early stage embryos were most difficult to culture through the 8- to 16-cell stage when grown in culture medium without oviducal cells. In Medium E %GS, 68% of the embryos cultured from the 1 2-cell stage halted division at the 8-cell stage, while only 8% reached 8 16-cells. Cleavage arrest was not related to cell death because embryos showed strong fluorescence after exposure to FDA. Furthermore, using Medium BM, only 5/16 embryos collected at 84 h after

6 ovulation (when most embryos are < 16-cells) developed beyond the 16-cell stage, whereas 18/21 collected at 96 h after ovulation (when most embryos are > 16-cells) developed to form blastocysts. In contrast, although the majority of embryos failed to develop past the 16-cell stage, cleavage arrest was not confined to a specific cell cycle in the simple chemically defined media tested, sug gesting that medium rather than stage of cell cycle was responsible for poor embryo development in vitro. The co-culture of 1-8-cell stage goat embryos with oviduct epithelial cells significantly enhanced the ability of goat embryos to be cultured in vitro when compared to culture medium alone. Co-culture with oviduct cells allowed a high percentage of embryos to avoid cleavage arrest at the 8-16-cell stage and maintained developmental rates equivalent to that observed in vivo up to the 4th cell cycle. The block that occurs during in-vitro culture of embryos has previously been overcome by oviduct epithelial co-culture systems in the sheep and cow (Eyestone et al, 1987; Gandolfi & Moor, 1987) and by the use of mouse oviduct organ expiant cultures in the mouse and hamster (Whittingham & Biggers, 1967; Minami et al, 1988). The mechanism by which epithelial cells exert their beneficial effect is not clear. Injection of cytoplasm of late 2-cell mouse embryos into 1-cell embryos leads to cleavage in inbred strains which classically block at the 2-cell stage in vitro (Muggleton-Harris et al, 1982), but whether the factor responsible is produced by the embryo or taken up from the surrounding environment by the embryo is not known. Several workers have reported the binding of oviducal proteins to embryos (Gaunt, 1985; St-Jacques & Bleau, 1988) or their presence in the perivitelline space (Kapur & Johnson, 1985). The positive effect exerted by the oviduct cells may be non-specific because a small percentage of sheep embryos can develop to form blastocysts when co-cultured with sheep fibroblasts (Gandolfi & Moor, 1987) and early bovine embryos develop through the 8-16-cell stage in culture with trophoblastic vesicles (Camous et al, 1984). Once embryos compact the oviduct co-culture system appears to lack the essential nutrients or conditions for the embryo to increase rapidly in cell number as observed in vivo. The failure of the oviduct co-culture system to maintain an effect in the cleavage rates of morulae is not surprising given that at the time of compaction embryos pass from the oviduct to the uterus where they are exposed to a different environment. Indeed, Lavranos & Seamark (1989) have shown that in the mouse a greater percentage of cultured 8-cell embryos reached the blastocyst stage and developed outgrowths in a uterine co-culture system than in medium alone. Fischer (1987) has also shown that supplementation with uterine flushings or culture media markedly promoted cell proliferation in rabbit blastocysts as measured by [3H]thymidine incorporation. In conjunction with the rapid increase in cell number after compaction, embryos also undergo various metabolic changes; for example, they begin to utilize glucose as an energy source and increase amino acid uptake (Epstein, 1975; Flood & Wiebold, 1988). Culture of post-compaction embryos in a simple medium such as BM permits similar development to the co-culture system and significantly better development than 10%GS. An amino acid or vitamin that is a more complex medium such as Medium E199 -Iinhibitory to embryo development, as with hypoxanthine which arrests mouse embryo develop ment at the 2-cell stage (Loutradis et al, 1987), may be present in the complex medium and removed by the oviduct cells. Our results further substantiate the conclusions of Gandolfi & Moor (1987) that early cleavagestage embryos have two major hurdles to overcome in vitro. Firstly, embryos must develop through the extended cell cycle where blockage is most likely to occur, and secondly, they must maintain developmental viability. To obtain an acceptable rate of development in vitro during prolonged culture requires further understanding of the factors controlling embryo cleavage and development in vivo and in vitro. We thank Dr A. O. Trounson for assistance with preparation of the manuscript; and Nicki Nash and Wayne Jones for their technical assistance. Most of this work was funded by Embryotechnology Ltd, and carried out at their research station at Rye, Victoria, Australia.

7 References Bavister, B.D. (1988) Role of oviductal secretions in embryonic growth in vivo and in vitro. Theriogenology 29, Bilton, R.J. & Moore, N.W. (1976) In vitro culture, storage and transfer of goat embryos. Aust. J. biol. Sci. 29, Cameron, A.W.N., Battye, K.M. & Trounson, A.O. (1988) Time of ovulation in goats (Capra hircus) induced to superovualte with PMSG. J. Reprod. Fert. 83, Camous, S., Heyman, Y., Meziou, W. & Menezo, Y. (1984) Cleavage beyond the block stage and survival after transfer of early bovine embryos cultured with trophoblastic vesicles. J. Reprod. Fert. 72, 479^85. Crosby, I.M., Gandolfi, F. & Moor, R.M. (1988) Control of protein synthesis during early cleavage of sheep embryos. J. Reprod. Feri. 82, Cummins, J.M., Breen, T.M., Harrison, K.L., Shaw, J.M., Wilson, L.M. & Hennessey, J.F. (1986) A formula for scoring human embryo growth rates in IVF: its value in predicting pregnancy and in com parison with visual elements of embryo quality. J. in vitro Fert. Emb. Trans. 3, Davis, D.L. (1985) Culture and storage of pig embryos. J. Reprod. Fert., Suppl 33, Davis, D.L. & Day, B.N. (1978) Cleavage and blastocyst formation by pig eggs in vitro. J. Anim. Sci. 46, Epstein, C.J. (1975) Gene expression and macromolecular synthesis during preimplantation embryonic development. Biol. Reprod. 12, Eyestone, W.H., Vignieri, J. & First, N.L. (1987) Coculture in early bovine embryos with oviductal epithelium. Theriogenology 27, 228 (abstr.). Fischer, B. (1987) Developmental retardation in cultured preimplantation rabbit embryos. J. Reprod. Fert. 79, Flood, M.R. & Wiebold, J.L. (1988) Glucose metabolism by preimplantation pig embryos. J. Reprod. Fert. 84, Gandolfi, F. & Moor, R.M. (1987) Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells. J. Reprod. Fert. 81, Gaunt, S.J. (1985) In vivo and in vitro cultured mouse preimplantation embryos differ in their display of a teratocarcinoma cell surface antigen: possible bind ing of an oviduct factor. J. Embryol. exp. Morph. 88, Goddard, M.J. & Pratt, H.M. (1983) Control of events during early cleavage of the mouse embryo: an analy sis of the "2 cell block". J. Embryol. exp. Morph. 73, Harlow, G.M. & Quinn, P. (1982) Development of preimplantation mouse embryos in vivo and in vitro. Aust. J. biol. Sci. 35, Kapur, R.P. & Johnson, L.V. (1985) An oviductal fluid glycoprotein associated with ovulated mouse ova and early embryos. Devi Biol. 112, Kola, L, Folb, P.L & Parker, M.L (1986) Maternal administration ofcyclophosphamide induces chromo somal abberations and inhibits cell number, histone synthesis and DNA synthesis in preimplantation mouse embryos. Teratog. Carcinog. Mutagen. 6, Lavranos, T.C. & Seamark, R.F. (1989) Addition of steroids to embryo-uterine monolayer co-culture enhances embryo survival and implantation in vitro. Reprod. Fértil. Devi 1, 41^16. Loutradis, D., John, D. & Kiessling, A.A. (1987) Hypoxanthine causes a 2-cell block in random bred mouse embryos. Biol. Reprod. 37, Minami, N., Bavister, B.D. & Intani. (1988) Develop ment of hamster two-cell embryos in the isolated mouse oviduct in organ culture system. Gamete Res. 19, Mohr, L.R. & Trounson, A.O. (1980) The use of fluorescein diacetate to assess embryo viability in the mouse. J. Reprod. Fert. 58, Moore, N.W. (1982) Liquid storage and culture of embryos of farm animals. In Embryo Transfer in Cattle, Sheep and Goats, pp EdsJ. N. Shelton, A. O. Trounson, N. W. Moore & J. W. James. Aust. Soc. Reprod. Biol., Union Offset. Com. Press, A.CT., Australia. Muggleton-Harris,., Whittingham, D. & Wilson, L. (1982) Cytoplasmic control of preimplantation devel opment in vitro in the mouse. Nature, Lond. 299, 460^461. Quinn, P., Barros, C. & Whittingham, D.G. (1982) Preservation of hamster oocytes to assay the fertiliz ing capacity of human spermatozoa. J. Reprod. Fert. 66, Quinn, P., Kerin, J.F. & Warnes, G.M. (1985) Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubai fluid. Fert. Steril. 44, 492^198. Salamonsen, L.A., Sum, O.W., Doughton, B. & Findlay, J. (1985) The effects of estrogen and progesterone in vivo on protein synthesis and secretion by cul tured epithelial cells from sheep endometrium. Endocrinology 117, St-Jacques, S. & Bleau, G. (1988) Monoclonal antibodies specific for an oviductal component associated with the hamster zona pellucida. J. Reprod. Immunol. 12, Tervit, H.R. & Rowson, L.E.A. (1974) Birth of lambs after culture of sheep ova in vitro for up to 6 days. J. Reprod. Fert. 38, Whittingham, D.G. & Biggers, J.D. (1967) Fallopian tube and early cleavage in the mouse. Nature, Lond. 213, Wright, R.W. & Bondioli, K.R. (1981) Aspects of in vitro fertilization and embryo culture in domestic animals. J. Anim. Sci. 53, Received 13 February 1989