Upsala J Med Sci 83: 135-139, 1978 Effect of the Endocrine State of Blastocyst Donors on the Time Required for Initiation of Tropholalast Outgrowth GRELS N R S L U N D and ORJAN LUNDKVIST From the Reproduction Research U n i t , Biomedicul Centre, Uppsala, Sweden ABSTRACT Blastocysts from mice in a state of delayed implantation af- ter ovariectomy were recovered on day 5 , 7 or 9 (day 1 was the day a vaginal plug was found). Blastocysts were also re- covered on day 7 from animals that had received an injection of oestradioLl7p 8, 16 or 24 hours earlier. The blastocysts were incubated in a modified Brinster’s medium to which serum had been added and the time of initiation of blasto- cyst outgrowth was recorded. Blastcysts from early delay similar to those occurring on blastocyst activation during implantation. Since the endocrine state of the host animal determines the activity of the blastocysts in vivo, it is also possible that the same state of the blastocyst donors determines the activ- ity of blastocysts in vitro. Knowledge of this matter is important for culture experiments. where the level of blastocyst activity is crucial. The aim of this study was therefore to test grew out before those from late delay. A steady state of outgrowth time was achieved on day 7. It is therefore sug- gested that blastocysts for culture experiments in which the metabolic activity level is crucial should not be recovered before the steady state is attained. Oestrogen injections caused earlier outgrowths, at least after 16 h, which indi- cates that the earlier outgrowth soon after ovariectomy might also he caused by a residual effect of the oestrogen. whether the interval between the start of the culture and the beginning of outgrowth of the blastocysts is to the endocrine state Of the blastocyst donors. Mouse blastocysts were recovered at dif- ferent times both during experimental delay of im- plantation and during oestrogen-induced activation for implantation. - a state of low metabolic activity (8, 1 1 , 19), and given subcutaneously daily, the day of flushing excluded show a characteristic ultrastructure ( 1 , 13). The depression of the blastocyst activity during delay probably depends both on the exogenous adminis- tration of progesterone (9) and on the declining in- fluence of ovarian oestrogen, since if oestrogen is given, the blastocysts will become activated and start to implant (7, 20). Mouse blastocysts in vitro will grow out on the bottom of the culture dish within a few days. This ( 1 ) . Blastocysts were recovered on day 5 , 7 or 9 during delay of implantation and on day 7-8, 16 or 24 h after a s. c. injection of 0.1 pg oestradiol-17P (AE Leo, Helsingborg, Sweden) dissolved in propylene glycol ( 1 pglml). The uteri were flushed with culture medium, that had previ- ously been equilibrated with 5 % CO, in air overnight. During the flushing procedure the medium was collected in Petri dishes containing liquid paraffin in order to main- tain the p H during the subsequent search for blastocysts. Brinster’s medium for ovum culture was used (2), in which process has been regarded as an analogue ofimplan- ultrastmctural (Naeslund, G. 8~ Nilsson, 0.: To be published) and metabolic (9, 15, 16, 18) changes the lactate had been replaced with 1.0 mg/ml of glUcose, which is appropriate from the &cell stage on in the mouse centration of 1 % was added to permit outgrowths (5). tation (6) and has been found to be associated with (3). Fetal calf serum (Flow, Ayrshire, Scotland) in a con- Immediately after flushing the blastocysts were trans- Unsalu J M r d Sc.i 83 136 G . N z s h n d and 0. Lundkvist ferred to 0.1-ml droplets of the medium under liquid paraf- fin by means of glass capillaries connected to an Agla micrometer syringe (Burroughs Wellcome & Co, London, England). The droplets were made the day before the culture started and were kept in plastic Petri dishes (Falcon, Oxnard, California, USA) at 37°C in an atmosphere of 5 % COP in air. Four to five blastocysts were placed in each droplet. Observations of the blastocysts were made with an in- verted microscope (Biovert, Reichert, Austria), bright- ground microscopy being used for screening purposes and phase contrast (magnification X160) to detect the first signs of outgrowth (Fig. ! and 2 ) . The initiation of the outgrowth was defined as the moment when growing cells were first seen outside the blastocyst contour. The ob- servations were made every 8 h and always by the same person. The first observation of an outgrowth was re- corded as occumng in the middle of the previous 8-hour interval. Analysis of variance was used to detect signifi- cant differences between the experimental groups. Fig. 1 . Blastocysts in culture drop- lets 24 h after explantation; day-7 group. The blastocysts are expanded without signs of trophoblast outgrowth. Bright field. X250. RESULTS The frequency distributions for the moment of initi- ation of blastocyst outgrowth are summarized in Fig. 3. The means for the groups, and the number of animals, blastocysts, and droplets are given in Table 1. The means for every droplet were used as observations in the subsequent tests of significance (Table 11). The duration of the delay influenced the moment of initiation of outgrowth, as the blastocysts from day 5 started to grow out 13 h earlier, on the, aver- age, than the blastocysts from day 7, a statistically significant difference, whereas the difference be- tween day-7 blastocysts and those from day 9 was non-significant. Of the blastocysts from day 5 , those encased in zona pellucida grew out earlier F i g . 2 . Outgrowing trophoblast cells around two blastocysts 64 h after explantation; day-7 group. Phase contras. ~ 2 5 0 . Upsalu J Med Sci 83 Effect of blastocyst donors on trophoblast outgrowth 137 Table I . Time required f o r initiation of outgrowth in vitro of blastocysts f r o m donors in various endocrine states Hours after initiation No. of of culture No. of blasto- No. of Group ( M + S .D.) animals cysts droplets Delay of implantation Day 5 Day 7 Day 9 4 4 f 9 19 33 8 57+ 10 40 97 21 61f 7 40 I18 24 Oestrogen activation Oestrogen 8 h 6 0 f 4 6 32 6 Oestrogen 24 h 9 f 4 8 29 6 Oestrogen 16 h 4 7 f 8 14 22 5 (mean 39 h) than the zona-free ones (mean 47 h). No tests of significance were performed, because of the small number of zona-encased blastocysts. Oestrogen injected into the donor mice had a D A Y 5 1 - 0 7 2 96 l2Oh 27 33 3 2 DAY 7 0 2 4 48 * 96 120 h 6 2 7 32 34 0 24 4 8 * 7 2 9’6 120 h D A Y 9 D A Y 9 2 7 32 34 2 9’6 120 h 1 OESTR 8 h , - I 0 2 4 4 8 * 7 2 9 6 120 h OESTR 16 h &*- 4 9 6 120 h 0 2 4 7 2 OESTR 2 4 h 4 8 72 96 120 h Fig. 3. Histograms representing the frequency distnbu- tions for the appearance of the first outgrowth. The figures within or above rectangles represent the number of blastocysts that started to grow out during each 8-hour interval. Arrows indicate means. Day 5: Blastocysts reco- vered on day 5 during delay of implantation.-Day 7: Blastocysts recovered on day 7 during delay of implanta- tion. Day 9: Blastocysts recovered on day 9 during delay of implantation. Oestrogen 8 h: Blastocysts recovered on day 7, 8 h after an oestrogen injection. Oestrogen 16 h: Blastocysts recovered on day 7, 16 h after an ostrogen in- jection. Oestrogen 24 h: Blastocysts recovered o n day 7 , 24 h after an oestrogen injection. marked effect on the blastocysts in vitro compared with those from day 7 of delay. No difference in the appearance of outgrowths was seen 8 h after the injection, whereas after 16 h there was a statistically significant difference. The effect was considerable 24 h after the injection, as the blastocysts grew out on the average 48 h earlier than the day-7 blasto- cysts from animals in a state of delay. DISCUSSION Blastocyst activity has often been determined by measuring specific metabolic steps, such as the in- corporation of RNA precursors (4, 12, 18) and of amino acids (16, 19) and the production of carbon dioxide (9, 14, 15). In the present experiment a new method was tried-determination of the time re- quired for initiation of trophoblastic outgrowth in vitro. This method supplements others by taking into account the proliferating capacity of tropho- blast cells. The technique is simple and easy to handle, which is an advantage when several ex- perimental groups are to be tested. The moment of initiation of blastocyst outgrowth was estimated by microscopic observations of the blastocyst cultures. However, the very first cells Table 11. Significance levels f o r the differences be- tween groups Group Significance level Day 5 vs. day 7 Day 7 vs. day 9 Day 7 vs. oestrogen 8 h Day 7 vs. oestrogen 16 h Day 7 vs. oestrogen 24 h PC0.005 Non-significant No apparent difference PC0.05 A manifest difference Upsala J Med Sci 83 138 G . N a l u n d and 0. Lundkvist growing out are difficult to distinguish, but as trophoblast cells proliferate rapidly, judging from the changes that occurred in the same outgrowth area between two observations, the influence of this error is probably of minor importance. The sensitiv- ity of the method depends upon the length of the in- terval between two observations. Too close obser- vations might disturb the growth of the blastocysts and in the present experiment an interval of 8 h was found suitable, which resulted in an accuracy of k 4 h for every single observation. The reliability of this method was judged to be adequate for the present purpose, as no statistically significant differences were observed between vari- ous batches of day-7 blastocysts cultured during different periods of the experiment. It is therefore concluded that the present technique of measuring the time required for blastocysts to start their out- growth in vitro can be useful for evaluating factors that influence the blastocyst activity. The results demonstrate that the activity of the blastocyst in vitro is, in fact, influenced by the endocrine state of the blastocyst donor. Thus, delayed blastocysts flushed soon after the ovariectomy grew out faster than those flushed a few days later. However, from day 7 onwards (i.e. after 4 days diapause) no difference were ob- served between the groups of delayed blastocysts (Table I). The conclusion may therefore be drawn that the slow attainment of a steady state of blasto- cyst ultrastructure (1, 13) and metabolic activity ( 1 1) during delay corresponds to a similar attain- ment of activity in vitro. When oestrogen was injected into the donor ani- mals, their blastocysts grew out faster in vitro. This effect was more marked the longer after the oestrogen injection the blastocysts were recovered. Further, blastocysts from day 5 of normal, non- delayed pregnancy will grow out faster than those from animals during delay of implantation (Naeslund, G . : Unpublished observation). Thus, if blastocysts are influenced by oestrogen before be- ing cultured the time required for initiation of out- growth in vitro will change. Therefore, in culture experiments where the level of blastocyst activity is crucial, it is important to define strictly the en- docrine state of the blastocyst donors and the time of recovery. Furthermore, if a basic level is desired, the present experiment suggests that delayed blastocysts should not be recovered earlier than day 7. U p s u h J M e d S c i 83 The outgrowth of trophoblast cells i n vitro'has been regarded as an analogue of the attachment and invasion of trophoblast cells in vivo (6). However, the blastocyst in vitro lives in a totally different environment and faces quite a different material for growth than the blastocyst in the uterine cavity. One possible way of determining whether the cells of the blastocyst respond differently to these two conditions is to compare the intracellular changes in vitro with those in v i v o . This kind of study requires well defined culture conditions to avoid irrelevant influences on the morphology. The present design for blastocyst culture seems suitable for this purpose and will be used in future experiments for ultrastructural comparisons of blastocyst develop- ments in vivo and in vitro (Naeslund, G. & Nilsson, 0.: To be published) and for testing some hypo- theses on the control of blastocyst growth. ACKNOWLEDGEMENT§ We thank Mrs Barbro Einarsson for skilful technical as- sistance and Dr Gunnar Ekbohm for statistic consultation. The study was financially supported by the Swedish Med- ical Research Council (Project No. 12X-70 to Professor Ove Nilsson) and the Swedish Foundation for Prenatal Research. REFERENCES 1. Bergstrom, S . : Delay of blastocyst implantation in the mouse by ovariectomy or lactation. A scanning electron microscope study. Fertil Steril23: 548, 1972. 2. Brinster, R. L.: A method for in vitro cultivation of mouse ova from two-cell to blastocyst. Exp Cell Res 32: 205, 1963. 3. Brinster, R. L . & Thomson, J. 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