Journal of in Vitro Fertilization and Embryo Transfer, Vol. 7, No. 5, 1990
Assessment of Hamster Blastocysts Derived from Eight-Cell Embryos Cultured in Hamster Embryo Culture Medium-2 (HECM-2): Cell Numbers and Viability Following Embryo Transfer POLANI B. SESHAGIRI 1'2 and BARRY D. BAVISTER 1
Submitted: January 27, 1990 Accepted: March 20, 1990
ture medium-2 could be useful for studies relating to embryo-derived proteins involved in the process of implantation and for the production of transgenic hamster embryos, for example, using embryonic stem cells.
The viability of hamster blastocysts, cultured from the eight-cell stage using hamster embryo culture medium-2, was examined by embryo transfer. Approximately 15-20 cultured hamster blastocysts were surgically transferred unilaterally to uterine horns of pseudopregnant recipient hamsters that had been mated to vasectomized males 3 days previously. Control recipients received in vivo developed, freshly recovered eight-cell embryos or blastocysts on day 2 or 3 of pseudopregnancy, respectively. Of the successful embryo transfers, the experimental group (receiving cultured blastocysts; n = 10) gave 51.8% implantations and 28.2% live pups. These values were 9closely similar to those of the controls; the percentages of control implantations and offspring were 51.1 and 34.0%, respectively, for eight-cell embryo transfer (n = 7, P > 0.69) and 48.5 and 28.9%for blastocyst transfer (n = 6, P > 0.52). To evaluate the quality of cultured hamster blastocysts, the following two parameters were examined. (1) The mean number of cells per blastocyst was 24.4 +- 0.7for cultured blastocysts. This value was similar to that (range, 14-24) obtained in this laboratory for in vivo developed freshly recovered blastocysts. (2) Oxygen consumption analysis revealed that cultured blastocysts actively respired at a level close to that observed with freshly recovered eight-cell embryos (slopes of oxygrams: 0.25 and 0.26, respectively). From these results, it is concluded that hamster blastocysts, cultured from the eightcell stage, are (a) qualitatively similar to freshly recovered in vivo developed blastocysts and (b) biologically viable as revealed by the production of live offspring upon embryo transfer. Therefore, hamster embryo cul-
KEY WORDS: hamster blastocysts; embryo culture; embryo
transfer; hamster embryo culture medium-2 (HECM-2).
INTRODUCTION The advent of technology for in vitro fertilization, followed by embryo culture and embryo transfer, offers the potential for making significant improvements in fecundity of economically important animals (1), as well as overcoming infertility in humans (2,3). However, further improvements have been difficult to achieve since the culture conditions for supporting normal development of mammalian embryos during preimplantation stages are still not optimal. During this phase of development, mammalian embryos are most vulnerable to alterations in the culture milieu and the physicochemical environments, as manifested in the quality and postimplantation viability of cultured embryos. The great majority of experiments on preimplantation embryo development is being carried out on laboratory animals, especially the mouse (4,5). In our laboratory, we have been using golden hamsters as a potentially useful alternative model for in vitro studies on the epigenetic regulation of preimplantation embryo development. It was discovered that the types of energy substrates provided in the culture medium largely regulate the development of hamster preimplantation embryos (6-11). Recently, by employing a chemically defined pro-
x Department of Veterinary Science, 1655 Linden Drive, University of Wisconsin, Madison, Wisconsin 53706. z To whom correspondence should be addressed. 229
0740-7769/90/1000-0229506.00/0 9 1990 Plenum Publishing Corporation
230
SESHAGIRI AND BAVISTER
tein-free culture medium containing 20 amino acids, namely, hamster embryo culture medium (HCEM)1, it was possible to overcome the developmental blocks characteristically observed in hamster embryos at the two- and four-cell stages (8,I1); eightcell embryos, cultured from the two-cell stage, gave viable offspring after embryo transfer (12). Using a similar medium (HECM-2) containing only four amino acids, we were able to increase markedly the proportion of blastocysts (>190.0%) cultured from the eight-cell stage (9,10). This new achievement is a distinct improvement over an early study in which only 60% blastocysts were obtained by culture of eight-cell hamster embryos and the viability of these blastocysts was minimal (13). Although the modified culture medium formulation (HECM-2) supports the development of large numbers of eightcell embryos to late blastocysts (14), the quality and viability of these blastocysts have not been evaluated. The present study provides several lines of evidence to show that hamster blastocysts cultured in HECM-2 are (i) qualitatively similar to in vivo developed freshly recovered blastocysts by comparison of mean number of cells, (ii) comparably metabolically active as shown by oxygen consumption analysis, and (iii) biologically viable as revealed by the production of live offspring after transfer of blastocysts to pseudopregnant recipients.
MATERIALS AND METHODS
Animals Golden hamsters (Mesocricetus auratus) from our laboratory-bred colony were kept on a lighting regimen of 14L: 10D (lights on at 0600). Experimental females, exhibiting synchronised 4-day estrous cycles, were normally housed in groups of four. Vasectomized males were used to induce pseudopregnancy. Vasectomies were performed on peripubertal males by double ligation and removal of an approximately 1.0-cm segment of the vas deferens. Before using for experiments, infertility of these males was checked by three consecutive matings confirming that the postovulatory vaginal discharge of mated females was devoid of sperm. As a further precaution against accidental impregnation, embryos were transferred only to one uterine horn of each recipient female. Alternate left and right uter-" ine horns of the recipients were used. Day 2 or 3 pseudopregnant recipient hamsters were used for
transfer of freshly recovered eight-cell embryos or of cultured/noncultured blastocysts, respectively. The day of postestrus discharge was designated day 1 (15).
Superovulation and Embryo Culture Female hamsters weighing approximately 100 g and having normal estrous cyclicity were superovulated by intraperitoneal (i.p.) injection of 15 IU of pregnant mare's serum gonadotropin (PMSG; Organon Inc., West Orange, NJ) at 1000h on the day of their postestrus discharge and mated to proven fertile males 3 days later (16). Pseudopregnant recipients were obtained by mating female hamsters to vasectomized males. Vaginal smears were performed on all mated females to ensure spermpositive donors and sperm-negative recipients. Donors were euthanized by cervical dislocation and their reproductive tracts were removed on day 3 or 4 of pregnancy for obtaining eight-cell embryos or blastocysts, respectively. The protocols for flushing and for culturing embryos were as described previously (9,10). The medium HECM-2 used for embryo culture was a glucose-, phosphate-, and protein-free, chemically defined medium containing Phe, Ile, Met, and Gin, 0.2 mM pyruvate, and 10 mM lactate as energy substrates (10). Hamster blastocysts, cultured from the eight-cell stage, were classified as early and late blastocysts according to the relative volume of the blastocoel cavity, i.e., 2/3 of the total embryo volume, respectively (13,14).
Transfer Procedure Recipient female hamsters were anesthetized to effect by i.p. injection of approximately 0.15 ml/100 g body weight of sodium pentobarbital (Nembutal, Abbott Laboratories, Chicago, IL). Animals were prepared for either left or right paralumbar incisions on the dorsal surface about 2 cm lateral to the vertebral column. The upper one-third of the uterine horn was pulled out from the abdominal cavity, and the fat pad surrounding the ovary was exposed and held retracted with the aid of hemostatic forceps. Sterile, warmed isotonic saline was applied periodically throughout the surgery to prevent drying of organs. A fiber-optics light source was used for illuminating the surgical field. Embryo transfer was performed using a dissecting microscope. With the help of an ultrafine watchmaker's forceps (No. 5),
Journal of in Vitro Fertilization and Embryo Transfer, Vol. 7, No. 5, 1990
VIABILITY OF CULTURED HAMSTER BLASTOCYSTS
the uterus receiving embryos was held in a suitable position for embryo transfer. Embryo aspiration and transfer were performed with a Pasteur pipet which had been pulled to an internal diameter of about 200 Ixm, attached to a mouth tube containing a sterile cotton filter plug. The tip was pierced through the uterotubal junction and placed into the uterine lumen. Approximately 15-20 embryos were deposited in a minimum volume of collection medium (~60) in the medium.
Nuclei Staining Procedure In order to determine the average number of nuclei per embryo (approximates mean number of
231
cells per blastocyst), cultured blastocysts were fixed and processed with a rapid whole-mount staining procedure using fluorescent dye Hoechst 33342 (HO) according to the procedure of Pursel et al. (17). Briefly, embryos were fixed in phosphatebuffered saline containing 1 mg/ml polyvinylalcohol and 1% formaldehyde. The stained embryos were mounted on a siliconized glass slide using a solution of 1% n-propylgallate contained in a mixture of 2.3% sodium citrate and glycerol (1:9) to help in reducing background fluorescence. Stained nuclei were counted using epifluorescence equipment: an inverted Diaphot microscope (Nikon) with a UVcube filter, a 400-nm dichroic mirror, Bp 340- to 380-nm filter, LP 430-nm filter, and UV source. Blastomeres were counted with a 40 x fluorescence objective.
Statistical Analysis The data from the experimental group (receiving cultured blastocysts), consisting of percentages of implantation sites and live pups born, were compared with each of the control values by two-sample t test (assuming unequal group variances) on the arcsin transformed values of the square roots of the proportions (18).
RESULTS Hamster blastocysts, cultured from the eight-cell stage in HECM-2, were transferred to uterine horns of asynchronous recipients on day 3 of pseudopregnancy in order to determine the ability of the cultured blastocysts to implant and develop into fetuses. About 51.8% of the transferred blastocysts implanted and began developing into fetuses, giving rise to 28.2% of live pups at term (Table I). Two control groups received either noncultured, freshly recovered eight-cell embryos (control-l) or blastocysts (control-2) on day 2 or 3 of pseudopregnancy, respectively. Control-1 gave 51.1% implantations and 34.0% pups and control-2 gave 48.5% implantations and 28.9% live pups, respectively. These control values were very similar to those obtained with the experimental group (Table I). Statistical analysis did not show any significant difference between the experimental group and control-1 (P > 0.69) or control-2 (P > 0.52). Almost all of the live pups delivered grew past weaning age analogous to the expected performance of the animals bred in our
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Table I. Development of Hamster Blastocysts Transferred to Pseudopregnant Recipients After Culturing from the Eight-Cell Stage in HECM-2
Group
Stage of embryos transferred a
Control-1
Fresh 8-cell
Control-2
Fresh blastocysts
Experimental
Blastocysts cultured f r o m 8-cell stage
No. of embryos transferred (No. of recipients)
No. of implantation sites (% -+ SE) b
No. of pups delivered (% -+ SE)
141 (7) 97 (6) 170 (10)
72 (51.1 +- 4.9) c,* 47 (48.5 -+ 5.3) d 88 (51.8 -+ 4.6) c,d
48 (34.0 -+ 6.3) e 28 (28.9 +- 3.9)r 48 (28.2 --- 3.2) eJ
a Hamster eight-cell embryos and blastocysts (fresh or cultured) were surgically transferred to pseudopregnant recipients on day 2 and 3, respectively. For details see text. b Number of implantations are the number of normal looking fetal inclusions visually observed at laparotomy on day 11 of pregnancy; the values do not include resorption sites. * Significance levels: P values were 0.69, 0.52, 0.82, and 0.8 for superscripts c, d, e, and f, respectively.
colony. Moreover, they were devoid of any detectable developmental/teratological abnormalities. Hamster blastocysts, cultured from the eight-cell stage in HECM-2, were subjected to Hoechst nuclei staining in order to determine the mean number of cells/blastocyst (Table II). The mean number of cells per blastocyst was relatively greater for cultured late blastocysts (24.4 -+ 0,7) than for noncultured in vivo developed freshly recovered blastocysts (range, 14-24; data from Bavister and Arzac, in preparation). Early blastocysts, derived from culture of eight-cell embryos in HECM-2, contained a mean number of cells/embryo (15.8 -+ 0.6) that was not different from that of the control blastocysts (Table II). Figure 1 documents the Hoechst-stained nuclei of late blastocysts cultured from the eightcell stage. Oxygen consumption analysis showed that the cultured blastocysts exhibited active respiratory acTable II. Comparison of Mean Number of Nuclei and Oxygen Consumption Values of Hamster Blastocysts Cultured from the Eight-Cell Stage and of Control Eight-Cell Embryos and Blastocysts Stage of embryos studied
No. of embryos stained a (No. of females)
Cultured early blastocysts Cultured late blastocysts Fresh blastocysts c Fresh 8-cell
46 (8) 70 (10) ---
Mean number of nuclei/embryo Slope -+ SE value b 15.8 --- 0.6
--
24.4 - 0.7
0.25
14-24 (range) --
-0.26
Embryos were stained using Hoechst staining by the method of Pursel et al. (17). b Oxygen consumption analysis was performed as detailed in the text; shown here is the slope value from the graphs in Fig. 2. c Data for periimplantation embryos from Bavister and Arzac (unpublished).
a
tivity. The pattern of oxygrams obtained for the cultured blastocysts was similar to that normally observed for the noncultured, freshly recovered eightcell embryos (Fig. 2) or blastocysts (data not shown). The slopes of these oxygrams gave values of 0.25 and 0.26 for cultured blastocysts and eightcell embryos, respectively (Table II).
DISCUSSION We have shown in this study that blastocysts, cultured from h a m s t e r eight-cell e m b r y o s in HECM-2 (10), were (a) of high quality judging by the mean number of cells per blastocyst, (b) metabolically active as shown by oxygen consumption analysis, and (c) biologically viable as confirmed by the production of normal offspring after surgical embryo transfer. The fact that a significant proportion of live pups was born (28.2%) upon transfer of cultured blastocysts (Table I) clearly proves the ability of HECM-2 to support normal development of hamster eight-cell embryos to blastocysts. This success rate of embryo transfer is approximately threefold higher than in a previous study in this laboratory which employed a protein-containing me, dium, namely, TALP (13). Therefore, protein supplementation of the culture medium is not required for full embryonic viability and may sometimes be detrimental to hamster embryos (McKiernan alad Bavister, in preparation) similar to that reported for mouse embryos (19). We conclude that the chemically defined protein-free culture medium, HECM-2 (10), used in this study represents an improved formulation for culturing late stages of hamster preimplantation embryos.
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VIABILITY OF CULTURED HAMSTER BLASTOCYSTS
Left
Right
Fig. 1. Hoechst-stained fluorescence microscopic picture of nuclei of hamster late blastocysts obtained after culture of eight-cell embryos in HECM-2. Photographed under UV light at an optical magnification of x20. For details see text. In this picture the number of nuclei was 20 (left) and 30 (right) per blastocyst. Reduced 25% for reproduction.
The percentage (28.2%) of live pups born upon transfer of cultured hamster blastocysts was similar to that of the control groups (Table I) which received noncultured, freshly recovered eight-cell embryos (34.0%) or blastocysts (28.9%). It is interesting to note that in all groups of embryo transfers, the observed percentages (48.5-51.8%) of implantations (at laparotomy) are closely comparable (Table I). This particular finding clearly indicates that the cultured blastocysts, derived from the eight-cell stage, are able to elicit a normal decidual response when transferred to a recipient uterus and, therefore, are almost equally capable of implanting in a normal fashion and proceeding with fetal development in comparison with in vivo developed eightcell embryos or blastocysts, following embryo transfer. These data demonstrate that culture of hamster eight-cell embryos for 18 hr in a chemically defined medium is compatible with maintenance of viability. Among the successful embryo transfers of all groups, it was noticed that implantation and subsequent fetal development were reasonably good only when recipients were asynchronous by 1 day (day 3
for blastocysts and day 2 for eight-cell embryos), while synchronous recipients supported very poor or no fetal development (data not shown). This finding indicates that although the blastocyst may be ready to implant, it remains quiescent and waits until the functional receptivity of the hamster uterus is achieved. It is possible that, during this "waiting period," blastocysts may undergo a "readjustment phase" suitable for in vivo environment prior to implantation. Hence, the uterine horns of asynchronous (retarded by one day) females are suitable for embryo transfer. This uterus-embryo asynchrony is noticed not only in the case of hamsters as in the present study but also in other species such as the mouse (4), rat (20), sheep (21,22), rabbit (23), and cow (24). Under our experimental conditions, it was noticed that not all embryos transferred to recipient hamsters gave birth to live pups. Similar findings on early embryonic loss after transfer of preimplantation embryos have been observed in species such as the rat (20), mouse (4), and a few others (21). This fetal mortality resulting in the reduction of number of pups born uniformly in all groups as in the
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SESHAGIRI AND BAVISTER
859~5
8-Cells slope = 0.26
tO
c 65
,
Blastocysts t
i
o •
95,
t
85' slope = 0.25 75
65 0
i
i
i
i
i
10
20
30
40
50
Time (minutes) Fig. 2. Oxygen consumption analysis of hamster preimplantation embryos. A minimum of approximately 60 embryos at each of the desired stages was suspended in 30 ~1 of HECM-2 and the oxygen consumption profile was recorded for at least 50 min. (A) Freshly recovered (in vivo developed) eight-cell embryos. (B) Blastocysts cultured from the eight-cell stage.
present study (Table I) could be attributed to several factors such as (a) the exact time of uterine receptivity, (b) the time taken for recovery and transfer of embryos, (c) handling of embryos during transfer, (d) variability of recipient and donor females, (e) surgical trauma during laparotomy, and (f) the skill of the operator. Another possible cause for fetal mortality during the postimplantation period of embryo development could be interembryo competition at a localized microenvironment in the uterus. Therefore, there would be favorable development of certain dominant embryos at the expense of others. In the present study, the use of HECM-2 not only produced biologically viable blastocysts from hamster eight-cell embryos, as confirmed by the production of live offspring after embryo transfer (Table I), but also produced good-quality blastocysts as revealed by the mean number of cells/blastocyst. This value for cultured blastocysts was found to be somewhat higher than that obtained for in vivo developed, freshly recovered blastocysts (Bavister and Arzac, unpublished). This increase could presumably be due to prolonged culture of embryos in HECM-2, beyond the time when implantation would normally have begun in vivo.
In order to evaluate further the quality of cultured blastocysts, a stringent metabolic test, i.e., oxygen consumption analysis has been performed. It is interesting to note from the oxygen consumption analysis that the cultured hamster blastocysts showed a rate of respiration that was comparable to that of freshly recovered in vivo developed either eight-cell embryos (Fig. 2) or blastocysts. A similar analysis has been performed for single mouse blastocysts (25). This objective metabolic criterion for the assessment of embryo viability is an important one, and if suitably modified, it could be routinely employed as a noninvasive quality assessment test prior to embryo transfers in laboratory and domestic animals as well as in humans. The protein-free, chemically defined embryo culture medium HECM-2 used here will be useful for studies on protein secretion by hamster preimplantation embryos, since the "signal-to-noise ratio" of secreted proteins will be very high. Therefore, HECM-2 should enable one to determine precisely which protein signals are produced at defined times during hamster preimplantation embryogenesis by analyzing the spent medium. Additional advantages of using this medium are the consistent reproducibility of preparing the medium and the elimination of possible microbial contamination associated with serum-supplemented media. Improved and consistent development in vitro of hamster two-cell embryos to morulae and blastocysts using HECM-1 (8,14) and eight-cell embryos to late blastocysts using HECM-2 (10,14) could make the hamster a valuable new laboratory animal model for (a) studies associated with the molecular aspects of mammalian preimplantation embryonic development and the process of implantation and (b) quality-control testing in human IVF programs. In the latter situation, a sensitive assay is needed to assist in protocols for quality-control testing of water, culture media, and culture wares (26). The use of HECM-1 and -2 should also facilitate the production of transgenic hamster embryos (e.g., using embryonic stem cells). In addition, these media could prove valuable for in vitro production and investigation of embryos from economically profitable domestic species such as the cow, sheep, and pig or other important mammalian species including primates. ACKNOWLEDGMENTS We thank Susan H. McKiernan for excellent technical support during the course of this work,
Journal of in Vitro Fertilization and Embryo Transfer, Vol. 7, No. 5, 1990
VIABILITY OF CULTURED HAMSTERBLASTOCYSTS
D e n n i s H e i s e y for his a s s i s t a n c e w i t h statistical a n a l y s i s , a n d Dr. R i c h a r d T a s c a for his a d v i c e . W e are p a r t i c u l a r l y grateful to Dr. E r i c O v e r s t r o m for his i n v a l u a b l e a d v i c e c o n c e r n i n g the a p p a r a t u s for m e a s u r i n g o x y g e n c o n s u m p t i o n of e m b r y o s . This w o r k w a s d o n e as p a r t o f the N a t i o n a l C o o p e r a t i v e P r o g r a m o n N o n - H u m a n in Vitro F e r t i l i z a t i o n a n d Preimplantation Development and was funded by the N a t i o n a l I n s t i t u t e of Child H e a l t h a n d H u m a n Development, NIH, t h r o u g h c o o p e r a t i v e agreem e n t HD-22023.
REFERENCES 1. Iritani A: Current status of biotechnological studies in mammalian reproduction. Fertil Steril 1988;50:543-551 2. Jones GS: Update on in vitro fertilization. Endocrine Rev 1984;5:62-75 3. Wood C, McMaster R, Rennie G, Trounson A, Leeton J: Factors influencing pregnancy rates following in vitro fertilization and embryo transfer. Fertil Steril 1985;43:245-250 4. George MA, Doe BG: The influence of handling procedures during mouse oocyte and embryo recovery on viability and subsequent development in vitro. J Vitro Fert Embryo Transfer 1989;6:69-72 5. George AG, Braude PR, Johnson MH, Sweetnam DG: Quality control in the IVF laboratory: In vitro and in vivo development of mouse embryos is unaffected by the quality of water used in culture media. Hum Reprod 1989;4:826--831 6. Bavister BD: Studies on the developmental blocks in cultured hamster embryos. In The Mammalian Preimplantation Embryo: Regulation of Growth and Differentiation in Vitro, BD Bavister (ed). New York, Plenum Press, 1987, pp 21% 250 7. Bavister BD: Regulation ofhamster preimplantation embryo development in vitro by glucose and phosphate. In Early Embryo Development and Paracrine Relationships, S Heyner, L Wiley (eds). New York, Alan R. Liss, 1990, pp 7%96 8. Schini SA, Bavister BD: Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose. Biol Reprod 1988;39:1183-1192 9. Seshagiri PB, Bavister BD: Glucose inhibits development of hamster 8-cell embryos in vitro. Biol Reprod 1989;40:59% 606 10. Seshagiri PB, Bavister BD: Phosphate is required for glucose inhibition of development of hamster 8-cell embryos in vitro. Biol Reprod 1989;40:607-614
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11. Monis H, Bavister BD: Development of 4-cell hamster embryos to the blastocyst stage in vitro and its regulation by components of the culture milieu. Reprod Fertil Dev 1990;2:1-9 12. Schini SA, Bavister BD: Normal offspring produced after transfer of hamster embryos grown from 2- to 8-cells in vitro using a chemically defined culture medium. Theriogenol 1990;33:1255-1262 13. Bavister BD, Leibfried ML, Lieberman G: Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol Reprod 1983;28:235-247 14. Seshagiri PB, Bavister BD: Relative developmental abilities of hamster 2- and 8-cell embryos cultured in HECM-1 and -2. J Exp Zool (accepted) 15. Orsini MW: The external vaginal phenomena characterizing the stages of the estrous cycle, pregnancy, pseudopregnancy, lactation and the anestrous hamster. Proc Anita Care Panel 1964;11:193-206 16. Bavister BD: A consistently successful procedure for in vitro fertilization of golden hamster eggs. Gamete Res 1989; 23:13%158 17. Pursel VG, Wall RJ, Rexroad CE Jr, Hammer RE, Brinster RL: A rapid whole-mount staining procedure for nuclei of mammalian embryos. Theriogenol 1985;24:687-691 18. Snedecor GW, Cochran WG: Statistical Methods. Ames, Iowa State University Press, 1982, pp 290-291 19. Caro CM, Trounson A: The effect of protein on preimplantation mouse embryo development in vitro. J Vitro Fert Embryo Transfer 1984;1:183-187 20. Vanderhyden BC, Armstrong DT: Decreased embryonic survival of in vitro fertilized oocytes in rats is due to retardation of preimplantation development. J Reprod Fert 1988;83:851-857 21. Pope WF: Uterine asynchrony: A cause of embryonic loss. Biol Reprod 1988;39:99%1003 22. Rexroad CE, Powell AM: Asynchronous transfer improves development of sheep embryos co-cultured in serum-free medium. Biol Reprod 1989;40(Suppl 1):137 (No. 268) 23. Chang MC: Development and fate of transferred rabbit ova or blastocyst in relation to the ovulation time of recipients. J Exp Zool 1950;114:197-226 24. Rowson LEA, Moore RM, Lawson RAS: Fertility following egg transfer in the cow: Effect of method, medium and synchronization of oestrus. J Reprod Fert 1969;18:517-523 25. Overstrom E: In vitro assessment of blastocyst differentiation. In The Mammalian Preimplantation Embryo: Regulation of Growth and Differentiation in Vitro, BD Bavister (ed). New York, Plenum Press, 1987, pp 95-116 26. Bavister BD, Andrews JC: A rapid sperm motility bioassay procedure for quality-control testing of water and culture media. J Vitro Fert Embryo Transfer 1988;5:67-75
Journal o f in Vitro Fertilization and Embryo Transfer, Vol. 7, No. 5, 1990
Assessment of Hamster Blastocysts Derived from Eight-Cell Embryos Cultured in Hamster Embryo Culture Medium-2 (HECM-2): Cell Numbers and Viability Following Embryo Transfer POLANI B. SESHAGIRI 1'2 and BARRY D. BAVISTER 1
Submitted: January 27, 1990 Accepted: March 20, 1990
ture medium-2 could be useful for studies relating to embryo-derived proteins involved in the process of implantation and for the production of transgenic hamster embryos, for example, using embryonic stem cells.
The viability of hamster blastocysts, cultured from the eight-cell stage using hamster embryo culture medium-2, was examined by embryo transfer. Approximately 15-20 cultured hamster blastocysts were surgically transferred unilaterally to uterine horns of pseudopregnant recipient hamsters that had been mated to vasectomized males 3 days previously. Control recipients received in vivo developed, freshly recovered eight-cell embryos or blastocysts on day 2 or 3 of pseudopregnancy, respectively. Of the successful embryo transfers, the experimental group (receiving cultured blastocysts; n = 10) gave 51.8% implantations and 28.2% live pups. These values were 9closely similar to those of the controls; the percentages of control implantations and offspring were 51.1 and 34.0%, respectively, for eight-cell embryo transfer (n = 7, P > 0.69) and 48.5 and 28.9%for blastocyst transfer (n = 6, P > 0.52). To evaluate the quality of cultured hamster blastocysts, the following two parameters were examined. (1) The mean number of cells per blastocyst was 24.4 +- 0.7for cultured blastocysts. This value was similar to that (range, 14-24) obtained in this laboratory for in vivo developed freshly recovered blastocysts. (2) Oxygen consumption analysis revealed that cultured blastocysts actively respired at a level close to that observed with freshly recovered eight-cell embryos (slopes of oxygrams: 0.25 and 0.26, respectively). From these results, it is concluded that hamster blastocysts, cultured from the eightcell stage, are (a) qualitatively similar to freshly recovered in vivo developed blastocysts and (b) biologically viable as revealed by the production of live offspring upon embryo transfer. Therefore, hamster embryo cul-
KEY WORDS: hamster blastocysts; embryo culture; embryo
transfer; hamster embryo culture medium-2 (HECM-2).
INTRODUCTION The advent of technology for in vitro fertilization, followed by embryo culture and embryo transfer, offers the potential for making significant improvements in fecundity of economically important animals (1), as well as overcoming infertility in humans (2,3). However, further improvements have been difficult to achieve since the culture conditions for supporting normal development of mammalian embryos during preimplantation stages are still not optimal. During this phase of development, mammalian embryos are most vulnerable to alterations in the culture milieu and the physicochemical environments, as manifested in the quality and postimplantation viability of cultured embryos. The great majority of experiments on preimplantation embryo development is being carried out on laboratory animals, especially the mouse (4,5). In our laboratory, we have been using golden hamsters as a potentially useful alternative model for in vitro studies on the epigenetic regulation of preimplantation embryo development. It was discovered that the types of energy substrates provided in the culture medium largely regulate the development of hamster preimplantation embryos (6-11). Recently, by employing a chemically defined pro-
x Department of Veterinary Science, 1655 Linden Drive, University of Wisconsin, Madison, Wisconsin 53706. z To whom correspondence should be addressed. 229
0740-7769/90/1000-0229506.00/0 9 1990 Plenum Publishing Corporation
230
SESHAGIRI AND BAVISTER
tein-free culture medium containing 20 amino acids, namely, hamster embryo culture medium (HCEM)1, it was possible to overcome the developmental blocks characteristically observed in hamster embryos at the two- and four-cell stages (8,I1); eightcell embryos, cultured from the two-cell stage, gave viable offspring after embryo transfer (12). Using a similar medium (HECM-2) containing only four amino acids, we were able to increase markedly the proportion of blastocysts (>190.0%) cultured from the eight-cell stage (9,10). This new achievement is a distinct improvement over an early study in which only 60% blastocysts were obtained by culture of eight-cell hamster embryos and the viability of these blastocysts was minimal (13). Although the modified culture medium formulation (HECM-2) supports the development of large numbers of eightcell embryos to late blastocysts (14), the quality and viability of these blastocysts have not been evaluated. The present study provides several lines of evidence to show that hamster blastocysts cultured in HECM-2 are (i) qualitatively similar to in vivo developed freshly recovered blastocysts by comparison of mean number of cells, (ii) comparably metabolically active as shown by oxygen consumption analysis, and (iii) biologically viable as revealed by the production of live offspring after transfer of blastocysts to pseudopregnant recipients.
MATERIALS AND METHODS
Animals Golden hamsters (Mesocricetus auratus) from our laboratory-bred colony were kept on a lighting regimen of 14L: 10D (lights on at 0600). Experimental females, exhibiting synchronised 4-day estrous cycles, were normally housed in groups of four. Vasectomized males were used to induce pseudopregnancy. Vasectomies were performed on peripubertal males by double ligation and removal of an approximately 1.0-cm segment of the vas deferens. Before using for experiments, infertility of these males was checked by three consecutive matings confirming that the postovulatory vaginal discharge of mated females was devoid of sperm. As a further precaution against accidental impregnation, embryos were transferred only to one uterine horn of each recipient female. Alternate left and right uter-" ine horns of the recipients were used. Day 2 or 3 pseudopregnant recipient hamsters were used for
transfer of freshly recovered eight-cell embryos or of cultured/noncultured blastocysts, respectively. The day of postestrus discharge was designated day 1 (15).
Superovulation and Embryo Culture Female hamsters weighing approximately 100 g and having normal estrous cyclicity were superovulated by intraperitoneal (i.p.) injection of 15 IU of pregnant mare's serum gonadotropin (PMSG; Organon Inc., West Orange, NJ) at 1000h on the day of their postestrus discharge and mated to proven fertile males 3 days later (16). Pseudopregnant recipients were obtained by mating female hamsters to vasectomized males. Vaginal smears were performed on all mated females to ensure spermpositive donors and sperm-negative recipients. Donors were euthanized by cervical dislocation and their reproductive tracts were removed on day 3 or 4 of pregnancy for obtaining eight-cell embryos or blastocysts, respectively. The protocols for flushing and for culturing embryos were as described previously (9,10). The medium HECM-2 used for embryo culture was a glucose-, phosphate-, and protein-free, chemically defined medium containing Phe, Ile, Met, and Gin, 0.2 mM pyruvate, and 10 mM lactate as energy substrates (10). Hamster blastocysts, cultured from the eight-cell stage, were classified as early and late blastocysts according to the relative volume of the blastocoel cavity, i.e., 2/3 of the total embryo volume, respectively (13,14).
Transfer Procedure Recipient female hamsters were anesthetized to effect by i.p. injection of approximately 0.15 ml/100 g body weight of sodium pentobarbital (Nembutal, Abbott Laboratories, Chicago, IL). Animals were prepared for either left or right paralumbar incisions on the dorsal surface about 2 cm lateral to the vertebral column. The upper one-third of the uterine horn was pulled out from the abdominal cavity, and the fat pad surrounding the ovary was exposed and held retracted with the aid of hemostatic forceps. Sterile, warmed isotonic saline was applied periodically throughout the surgery to prevent drying of organs. A fiber-optics light source was used for illuminating the surgical field. Embryo transfer was performed using a dissecting microscope. With the help of an ultrafine watchmaker's forceps (No. 5),
Journal of in Vitro Fertilization and Embryo Transfer, Vol. 7, No. 5, 1990
VIABILITY OF CULTURED HAMSTER BLASTOCYSTS
the uterus receiving embryos was held in a suitable position for embryo transfer. Embryo aspiration and transfer were performed with a Pasteur pipet which had been pulled to an internal diameter of about 200 Ixm, attached to a mouth tube containing a sterile cotton filter plug. The tip was pierced through the uterotubal junction and placed into the uterine lumen. Approximately 15-20 embryos were deposited in a minimum volume of collection medium (~60) in the medium.
Nuclei Staining Procedure In order to determine the average number of nuclei per embryo (approximates mean number of
231
cells per blastocyst), cultured blastocysts were fixed and processed with a rapid whole-mount staining procedure using fluorescent dye Hoechst 33342 (HO) according to the procedure of Pursel et al. (17). Briefly, embryos were fixed in phosphatebuffered saline containing 1 mg/ml polyvinylalcohol and 1% formaldehyde. The stained embryos were mounted on a siliconized glass slide using a solution of 1% n-propylgallate contained in a mixture of 2.3% sodium citrate and glycerol (1:9) to help in reducing background fluorescence. Stained nuclei were counted using epifluorescence equipment: an inverted Diaphot microscope (Nikon) with a UVcube filter, a 400-nm dichroic mirror, Bp 340- to 380-nm filter, LP 430-nm filter, and UV source. Blastomeres were counted with a 40 x fluorescence objective.
Statistical Analysis The data from the experimental group (receiving cultured blastocysts), consisting of percentages of implantation sites and live pups born, were compared with each of the control values by two-sample t test (assuming unequal group variances) on the arcsin transformed values of the square roots of the proportions (18).
RESULTS Hamster blastocysts, cultured from the eight-cell stage in HECM-2, were transferred to uterine horns of asynchronous recipients on day 3 of pseudopregnancy in order to determine the ability of the cultured blastocysts to implant and develop into fetuses. About 51.8% of the transferred blastocysts implanted and began developing into fetuses, giving rise to 28.2% of live pups at term (Table I). Two control groups received either noncultured, freshly recovered eight-cell embryos (control-l) or blastocysts (control-2) on day 2 or 3 of pseudopregnancy, respectively. Control-1 gave 51.1% implantations and 34.0% pups and control-2 gave 48.5% implantations and 28.9% live pups, respectively. These control values were very similar to those obtained with the experimental group (Table I). Statistical analysis did not show any significant difference between the experimental group and control-1 (P > 0.69) or control-2 (P > 0.52). Almost all of the live pups delivered grew past weaning age analogous to the expected performance of the animals bred in our
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Table I. Development of Hamster Blastocysts Transferred to Pseudopregnant Recipients After Culturing from the Eight-Cell Stage in HECM-2
Group
Stage of embryos transferred a
Control-1
Fresh 8-cell
Control-2
Fresh blastocysts
Experimental
Blastocysts cultured f r o m 8-cell stage
No. of embryos transferred (No. of recipients)
No. of implantation sites (% -+ SE) b
No. of pups delivered (% -+ SE)
141 (7) 97 (6) 170 (10)
72 (51.1 +- 4.9) c,* 47 (48.5 -+ 5.3) d 88 (51.8 -+ 4.6) c,d
48 (34.0 -+ 6.3) e 28 (28.9 +- 3.9)r 48 (28.2 --- 3.2) eJ
a Hamster eight-cell embryos and blastocysts (fresh or cultured) were surgically transferred to pseudopregnant recipients on day 2 and 3, respectively. For details see text. b Number of implantations are the number of normal looking fetal inclusions visually observed at laparotomy on day 11 of pregnancy; the values do not include resorption sites. * Significance levels: P values were 0.69, 0.52, 0.82, and 0.8 for superscripts c, d, e, and f, respectively.
colony. Moreover, they were devoid of any detectable developmental/teratological abnormalities. Hamster blastocysts, cultured from the eight-cell stage in HECM-2, were subjected to Hoechst nuclei staining in order to determine the mean number of cells/blastocyst (Table II). The mean number of cells per blastocyst was relatively greater for cultured late blastocysts (24.4 -+ 0,7) than for noncultured in vivo developed freshly recovered blastocysts (range, 14-24; data from Bavister and Arzac, in preparation). Early blastocysts, derived from culture of eight-cell embryos in HECM-2, contained a mean number of cells/embryo (15.8 -+ 0.6) that was not different from that of the control blastocysts (Table II). Figure 1 documents the Hoechst-stained nuclei of late blastocysts cultured from the eightcell stage. Oxygen consumption analysis showed that the cultured blastocysts exhibited active respiratory acTable II. Comparison of Mean Number of Nuclei and Oxygen Consumption Values of Hamster Blastocysts Cultured from the Eight-Cell Stage and of Control Eight-Cell Embryos and Blastocysts Stage of embryos studied
No. of embryos stained a (No. of females)
Cultured early blastocysts Cultured late blastocysts Fresh blastocysts c Fresh 8-cell
46 (8) 70 (10) ---
Mean number of nuclei/embryo Slope -+ SE value b 15.8 --- 0.6
--
24.4 - 0.7
0.25
14-24 (range) --
-0.26
Embryos were stained using Hoechst staining by the method of Pursel et al. (17). b Oxygen consumption analysis was performed as detailed in the text; shown here is the slope value from the graphs in Fig. 2. c Data for periimplantation embryos from Bavister and Arzac (unpublished).
a
tivity. The pattern of oxygrams obtained for the cultured blastocysts was similar to that normally observed for the noncultured, freshly recovered eightcell embryos (Fig. 2) or blastocysts (data not shown). The slopes of these oxygrams gave values of 0.25 and 0.26 for cultured blastocysts and eightcell embryos, respectively (Table II).
DISCUSSION We have shown in this study that blastocysts, cultured from h a m s t e r eight-cell e m b r y o s in HECM-2 (10), were (a) of high quality judging by the mean number of cells per blastocyst, (b) metabolically active as shown by oxygen consumption analysis, and (c) biologically viable as confirmed by the production of normal offspring after surgical embryo transfer. The fact that a significant proportion of live pups was born (28.2%) upon transfer of cultured blastocysts (Table I) clearly proves the ability of HECM-2 to support normal development of hamster eight-cell embryos to blastocysts. This success rate of embryo transfer is approximately threefold higher than in a previous study in this laboratory which employed a protein-containing me, dium, namely, TALP (13). Therefore, protein supplementation of the culture medium is not required for full embryonic viability and may sometimes be detrimental to hamster embryos (McKiernan alad Bavister, in preparation) similar to that reported for mouse embryos (19). We conclude that the chemically defined protein-free culture medium, HECM-2 (10), used in this study represents an improved formulation for culturing late stages of hamster preimplantation embryos.
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VIABILITY OF CULTURED HAMSTER BLASTOCYSTS
Left
Right
Fig. 1. Hoechst-stained fluorescence microscopic picture of nuclei of hamster late blastocysts obtained after culture of eight-cell embryos in HECM-2. Photographed under UV light at an optical magnification of x20. For details see text. In this picture the number of nuclei was 20 (left) and 30 (right) per blastocyst. Reduced 25% for reproduction.
The percentage (28.2%) of live pups born upon transfer of cultured hamster blastocysts was similar to that of the control groups (Table I) which received noncultured, freshly recovered eight-cell embryos (34.0%) or blastocysts (28.9%). It is interesting to note that in all groups of embryo transfers, the observed percentages (48.5-51.8%) of implantations (at laparotomy) are closely comparable (Table I). This particular finding clearly indicates that the cultured blastocysts, derived from the eight-cell stage, are able to elicit a normal decidual response when transferred to a recipient uterus and, therefore, are almost equally capable of implanting in a normal fashion and proceeding with fetal development in comparison with in vivo developed eightcell embryos or blastocysts, following embryo transfer. These data demonstrate that culture of hamster eight-cell embryos for 18 hr in a chemically defined medium is compatible with maintenance of viability. Among the successful embryo transfers of all groups, it was noticed that implantation and subsequent fetal development were reasonably good only when recipients were asynchronous by 1 day (day 3
for blastocysts and day 2 for eight-cell embryos), while synchronous recipients supported very poor or no fetal development (data not shown). This finding indicates that although the blastocyst may be ready to implant, it remains quiescent and waits until the functional receptivity of the hamster uterus is achieved. It is possible that, during this "waiting period," blastocysts may undergo a "readjustment phase" suitable for in vivo environment prior to implantation. Hence, the uterine horns of asynchronous (retarded by one day) females are suitable for embryo transfer. This uterus-embryo asynchrony is noticed not only in the case of hamsters as in the present study but also in other species such as the mouse (4), rat (20), sheep (21,22), rabbit (23), and cow (24). Under our experimental conditions, it was noticed that not all embryos transferred to recipient hamsters gave birth to live pups. Similar findings on early embryonic loss after transfer of preimplantation embryos have been observed in species such as the rat (20), mouse (4), and a few others (21). This fetal mortality resulting in the reduction of number of pups born uniformly in all groups as in the
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SESHAGIRI AND BAVISTER
859~5
8-Cells slope = 0.26
tO
c 65
,
Blastocysts t
i
o •
95,
t
85' slope = 0.25 75
65 0
i
i
i
i
i
10
20
30
40
50
Time (minutes) Fig. 2. Oxygen consumption analysis of hamster preimplantation embryos. A minimum of approximately 60 embryos at each of the desired stages was suspended in 30 ~1 of HECM-2 and the oxygen consumption profile was recorded for at least 50 min. (A) Freshly recovered (in vivo developed) eight-cell embryos. (B) Blastocysts cultured from the eight-cell stage.
present study (Table I) could be attributed to several factors such as (a) the exact time of uterine receptivity, (b) the time taken for recovery and transfer of embryos, (c) handling of embryos during transfer, (d) variability of recipient and donor females, (e) surgical trauma during laparotomy, and (f) the skill of the operator. Another possible cause for fetal mortality during the postimplantation period of embryo development could be interembryo competition at a localized microenvironment in the uterus. Therefore, there would be favorable development of certain dominant embryos at the expense of others. In the present study, the use of HECM-2 not only produced biologically viable blastocysts from hamster eight-cell embryos, as confirmed by the production of live offspring after embryo transfer (Table I), but also produced good-quality blastocysts as revealed by the mean number of cells/blastocyst. This value for cultured blastocysts was found to be somewhat higher than that obtained for in vivo developed, freshly recovered blastocysts (Bavister and Arzac, unpublished). This increase could presumably be due to prolonged culture of embryos in HECM-2, beyond the time when implantation would normally have begun in vivo.
In order to evaluate further the quality of cultured blastocysts, a stringent metabolic test, i.e., oxygen consumption analysis has been performed. It is interesting to note from the oxygen consumption analysis that the cultured hamster blastocysts showed a rate of respiration that was comparable to that of freshly recovered in vivo developed either eight-cell embryos (Fig. 2) or blastocysts. A similar analysis has been performed for single mouse blastocysts (25). This objective metabolic criterion for the assessment of embryo viability is an important one, and if suitably modified, it could be routinely employed as a noninvasive quality assessment test prior to embryo transfers in laboratory and domestic animals as well as in humans. The protein-free, chemically defined embryo culture medium HECM-2 used here will be useful for studies on protein secretion by hamster preimplantation embryos, since the "signal-to-noise ratio" of secreted proteins will be very high. Therefore, HECM-2 should enable one to determine precisely which protein signals are produced at defined times during hamster preimplantation embryogenesis by analyzing the spent medium. Additional advantages of using this medium are the consistent reproducibility of preparing the medium and the elimination of possible microbial contamination associated with serum-supplemented media. Improved and consistent development in vitro of hamster two-cell embryos to morulae and blastocysts using HECM-1 (8,14) and eight-cell embryos to late blastocysts using HECM-2 (10,14) could make the hamster a valuable new laboratory animal model for (a) studies associated with the molecular aspects of mammalian preimplantation embryonic development and the process of implantation and (b) quality-control testing in human IVF programs. In the latter situation, a sensitive assay is needed to assist in protocols for quality-control testing of water, culture media, and culture wares (26). The use of HECM-1 and -2 should also facilitate the production of transgenic hamster embryos (e.g., using embryonic stem cells). In addition, these media could prove valuable for in vitro production and investigation of embryos from economically profitable domestic species such as the cow, sheep, and pig or other important mammalian species including primates. ACKNOWLEDGMENTS We thank Susan H. McKiernan for excellent technical support during the course of this work,
Journal of in Vitro Fertilization and Embryo Transfer, Vol. 7, No. 5, 1990
VIABILITY OF CULTURED HAMSTERBLASTOCYSTS
D e n n i s H e i s e y for his a s s i s t a n c e w i t h statistical a n a l y s i s , a n d Dr. R i c h a r d T a s c a for his a d v i c e . W e are p a r t i c u l a r l y grateful to Dr. E r i c O v e r s t r o m for his i n v a l u a b l e a d v i c e c o n c e r n i n g the a p p a r a t u s for m e a s u r i n g o x y g e n c o n s u m p t i o n of e m b r y o s . This w o r k w a s d o n e as p a r t o f the N a t i o n a l C o o p e r a t i v e P r o g r a m o n N o n - H u m a n in Vitro F e r t i l i z a t i o n a n d Preimplantation Development and was funded by the N a t i o n a l I n s t i t u t e of Child H e a l t h a n d H u m a n Development, NIH, t h r o u g h c o o p e r a t i v e agreem e n t HD-22023.
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11. Monis H, Bavister BD: Development of 4-cell hamster embryos to the blastocyst stage in vitro and its regulation by components of the culture milieu. Reprod Fertil Dev 1990;2:1-9 12. Schini SA, Bavister BD: Normal offspring produced after transfer of hamster embryos grown from 2- to 8-cells in vitro using a chemically defined culture medium. Theriogenol 1990;33:1255-1262 13. Bavister BD, Leibfried ML, Lieberman G: Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol Reprod 1983;28:235-247 14. Seshagiri PB, Bavister BD: Relative developmental abilities of hamster 2- and 8-cell embryos cultured in HECM-1 and -2. J Exp Zool (accepted) 15. Orsini MW: The external vaginal phenomena characterizing the stages of the estrous cycle, pregnancy, pseudopregnancy, lactation and the anestrous hamster. Proc Anita Care Panel 1964;11:193-206 16. Bavister BD: A consistently successful procedure for in vitro fertilization of golden hamster eggs. Gamete Res 1989; 23:13%158 17. Pursel VG, Wall RJ, Rexroad CE Jr, Hammer RE, Brinster RL: A rapid whole-mount staining procedure for nuclei of mammalian embryos. Theriogenol 1985;24:687-691 18. Snedecor GW, Cochran WG: Statistical Methods. Ames, Iowa State University Press, 1982, pp 290-291 19. Caro CM, Trounson A: The effect of protein on preimplantation mouse embryo development in vitro. J Vitro Fert Embryo Transfer 1984;1:183-187 20. Vanderhyden BC, Armstrong DT: Decreased embryonic survival of in vitro fertilized oocytes in rats is due to retardation of preimplantation development. J Reprod Fert 1988;83:851-857 21. Pope WF: Uterine asynchrony: A cause of embryonic loss. Biol Reprod 1988;39:99%1003 22. Rexroad CE, Powell AM: Asynchronous transfer improves development of sheep embryos co-cultured in serum-free medium. Biol Reprod 1989;40(Suppl 1):137 (No. 268) 23. Chang MC: Development and fate of transferred rabbit ova or blastocyst in relation to the ovulation time of recipients. J Exp Zool 1950;114:197-226 24. Rowson LEA, Moore RM, Lawson RAS: Fertility following egg transfer in the cow: Effect of method, medium and synchronization of oestrus. J Reprod Fert 1969;18:517-523 25. Overstrom E: In vitro assessment of blastocyst differentiation. In The Mammalian Preimplantation Embryo: Regulation of Growth and Differentiation in Vitro, BD Bavister (ed). New York, Plenum Press, 1987, pp 95-116 26. Bavister BD, Andrews JC: A rapid sperm motility bioassay procedure for quality-control testing of water and culture media. J Vitro Fert Embryo Transfer 1988;5:67-75
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