MOLECULAR REPRODUCTION AND DEVELOPMENT 27:224-229 (1990)

Early Embryo Development in the Siberian Hamster (Phodopus sungorus) GARY L. NIEDER AND TERRI L. CAPRI0 Department of Anatomy, Wright State University School of Medicine, Dayton, Ohio

ABSTRACT Development of preimplantation embryos of the Siberian hamster (Phodopus sungorus] in vivo and in vitro was examined. The timing of early development in vivo was found to be slower than that reported for the golden hamster. Progression through the cleavage stages, cavitation, and hatching from the zona pellucida occurred later, with blastocyst formation beginning on the afternoon of day 4 and uterine attachment occurring early on day 5. In vitro, morulae, and early blastocysts collected on day 4 and cultured in serum-containing medium formed expanded blastocysts and some began to hatch from the zona pellucida. With extended culture, blastocysts attached and formed trophoblast outgrowths. Outgrowth was characterized by an initial migration of small cells from the blastocyst, followed by formation of a sheet of trophoblast giant cells. Differences in the morphology of outgrowth between the hamster and mouse suggest that further comparative studies with the Siberian hamster may be useful. Key Words: Preimplantation embryo, Blastocyst, Trophoblast

ever, the steroid hormone profile and pattern of follicular development during the cycle are significantly different from other short estrus cycle species including the golden hamster (Wynne-Edwards et al., 1987). These differences in reproductive physiology between Siberian and golden hamsters may extend to the timing of pre- and peri-implantation events and mechanism of implantation. The purpose of this study was to establish the time course of normal preimplantation development in the Siberian hamster and to determine if embryos could be cultured to form blastocyst outgrowths in vitro using a system developed for the mouse.

MATERIALS AND METHODS Materials Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s Modified Eagle’s Medium with 4.5 mg glucose/ ml (DMEM), endotoxin screened tissue culture grade water, penicillin/streptomycin mixture, and fetal calf serum (FCS) were obtained from Gibco (Grand Island, NY). Fraction V bovine serum albumin (BSA), glutamine, and sodium pyruvate were from Sigma (St. Louis, MO). Flat-bottom, 96-well tissue culture plates were from Corning (Corning, NY).

INTRODUCTION

Animals

Early embryonic development and implantation have been described in most laboratory species and domestic animals. It is evident from comparative studies that there is considerable variation between species in the timing of preimplantation development and mechanisms of implantation (Wimsatt, 1975; Weitlauf, 1988). The Siberian or Djungarian hamster (Phodopus sungorus), a dwarf hamster native to Northern China and the Soviet Union, has become a useful model in the study of photoperiodic mechanisms controlling the male reproductive system and pelage color (Yellon and Goldman, 1984; Stetson et al., 1989). Although this species has been propagated in laboratory colonies for many years, little is known about the female reproductive cycle and early embryo development. As in the golden hamster, a 4-day estrus cycle has been described based on cyclic changes in either vaginal smear morphology or receptivity to the male (Pilborough, 1971; Iakovenko, 1974; Wynne-Edwards et al., 1987). How-

A colony of Siberian hamsters has been maintained a t this institution since 1979. A 14 h r 1ight:lO h r dark photoperiod (lights on from 06.30 to 20.30 hr) was maintained; lab chow and water were available ad libitum. Sexually mature virgin females were paired singly with males, and on each successive morning vaginal smears, collected by 20 p1 saline lavage, were examined for the presence of sperm. Although a seminal vaginal plug similar to that found in mice was sometimes found in mated animals, this was often lost by the time animals were examined in the morning since, in most cases, no plug was found in females hav-

0 1990 WILEY-LISS, INC.

Received January 23, 1990; accepted May 21, 1990. Address reprint requests to Dr. G.L. Nieder, Department of Anatomy, Wright State University School of Medicine, Dayton, OH 45435.

SIBERIAN HAMSTER EMBRYOS

225

TABLE 1. Recovery of Siberian Hamster Embryos During the Preimplantation Period of Pregnancy Stage of development Time of collection" Day 2 (10.0015.00 hr) Day 3 (12.0015.00 hr) Day 4 (10.0015.00 hr) Day 4 (20.0023.00 hr) Day 5 (8.0011.00 hr)

Number of animals

Embryos recovered

Embryos/ animal (mean 2 S.E.M.)

2cell

3-4cell

5-8cell

Morula

Unhatched blastocvsts

Hatched blastocvsts

13

59

4.54

2

0.47

59

0

0

0

0

0

25

116

4.64

Ifr

0.37

7

40

69

0

0

0

14

57

4.07

2

0.53

0

1

1

16

39

0

7

33

4.71 t 0.56

0

0

1

2

20

10

7

1

0.14 t 0.13

0

0

0

1

0

0

"Day 1 is defined as the day of a sperm-positive vaginal smear.

Fig. 1. Preimplantation development of Siberian hamster embryos in vivo. Embryos were flushed from the oviducts or uteri of timed pregnant animals. Embryos were a t the two-cell stage on day 2 (a), the four- to eight-cell stage on day 3 (b),morula or early blastocyst

stage on the afternoon of day 4 (c), and at the unhatched (d) or hatched (e)blastocyst stage late on day 4. Immediately after flushing, all blastocysts had a small blastocoel and well demarcated inner cell mass. Hoffman modulation contrast optics; bar = 100 pm.

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G.L. NIEDER AND T.L. CAPRI0

Fig. 2. Culture of day 4 morulae and early blastocysts. Day 4 embryos cultured in DMEM with FCS for 24 hr (a) and 72 hr (b). Within a few hours in vitro, blastocoels expanded from their small volume seen when removed from the uterus (compare a to Fig. Id). Initiation of hatching is seen in (b). Bar = 100 wm.

ing a sperm-positive smear. After the sperm positive day, designated day 1of pregnancy, mating pairs were left undisturbed until the time of embryo collection.

Embryo Collection and Culture At specified times, animals were sacrificed and oviducts and uteri were flushed with DPBS containing 0.1 mg/ml BSA. The location and stage of development of all embryos found were recorded. For culture experiments, Siberian hamster embryos were collected between 12.00 h r and 14.00 h r of day 4 (morula and early blastocyst stage) or between 21.00 h r and 23.00 h r (expanded hatched and unhatched blastocyst stage). Embryos were pooled and cultured in DMEM supplemented with 4 m g BSA/ml, 10% FCS, 292 pg glutamine/ml, 56 pg sodium pyruvate/ml, 100 U penicillin/ml, and 100 pg streptomycin/ml. This medium supports hatching, attachment, and outgrowth of mouse blastocysts (Nieder, 1990). Cultures were carried out in 200 p1 volumes using 96-well tissue culture plates, at 37°C under a humidified 5% CO, atmosphere. For comparison, day 4 blastocysts from naturally mated ICR Swiss albino mice (Harlan, Indianapolis, IN) were collected and cultured under identical conditions.

RESULTS Timing of Early Development Embryos were collected a t specified times during gestation and their location and developmental stage noted (Table 1, Fig. 1).On the morning of day 1, onecell embryos were found in the ampullar portion of the oviduct and by that afternoon had begun shedding the cumulus. Two-cell embryos were found in the oviduct

on the afternoon of day 2, while four-cell to eight-cell stages were found on day 3. Development was asynchronous by day 3 since within individual animals embryos with from four to eight blastomeres were recovered. On the afternoon of day 4 (12.00-15.00 hr), morulae and early blastocysts were recovered from the uterus, while late on day 4 (20.00-23.00 hr) both unhatched (zona-encased) and hatched blastocysts were found. Both hatched and unhatched blastocysts had small blastocoels and well-defined inner cell masses. Hatching and attachment were apparently complete by 08.00 h r of day 5 since embryos could no longer be flushed out of the uterus and by 12.00 hr, decidual sites could easily be seen. There were no significant differences (P>.05 in unpaired t test) between the mean number of embryos at each preimplantation stage and the mean litter size in our colony (3.74 pupdlitter), implying minimal postimplantation embryo loss. Also there was no significant difference in the mean number of embryos recovered from the right and left sides of the reproductive tract, although in a few cases all embryos were found in one side.

Embryo Culture In vitro development of day 4 morula and early blastocysts was supported by DMEM with FCS. Nine of eleven day 4 morulae and eleven of eleven early blastocysts cultured in this medium completed expansion and a few expanded blastocysts (one cultured from the morula stage and four from the early blastocyst stage) began to hatch (Fig. 2). The blastocyst shown in Figure 2b hatched completely from the zona, but in most cases hatching only proceeded to the extrusion of a few processes through the zona before degeneration after 3-4

Fig. 3. Blastocyst attachment and outgrowth in vitro. An unhatched blastocyst was collected a t 21.00 hr of day 4 and cultured in DMEM with FCS. Hatching, blastocyst attachment, and migration of a population of small cells was evident by 63 hr of culture (a).Continued division of these migrating cells was evident at 67 hr (b). By 83 hr ( c ) ,a sheet of spreading trophoblast was seen

which continued to spread a t 108 (d),114 (e),132 (0, and 160 hr (g). Under identical conditions, a d a y 4 mouse blastocyst hatched and began outgrowth within 48 hr and by 72 hr had formed an extensive outgrowth (h).Bar = 100 Fm.

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G.L. NIEDER AND T.L. C A P R I 0

A culture system that has reliably supported mouse blastocyst attachment and outgrowth (Nieder, 1990) was found to also support Siberian hamster blastocyst development. Although the ability of the hamster blastocyst to hatch in vitro appears less than that of the mouse blastocyst, once free of the zona pellucida trophoblast outgrowth occurred with high frequency. In contrast to our observations in mice, outgrowth of the Siberian hamster embryos in vitro was delayed under our culture conditions with a 2 to 3 day lag before trophoblast spreading is seen. Another difference between mice and the Siberian hamster is the group of small dividing cells which were always seen to initially migrate from the hamster embryo. This type of proliferation was not typically seen in mouse blastocyst outgrowths and may represent a population of very invasive trophoblast cells in the hamster. Also the trophoblast giant cells in the Siberian hamster are larger but fewer in number than found in mouse outgrowths. The significance of the observed morphological variations of blastocyst outgrowth between rodent species is not known but may reflect differences in their mechanisms of implantation. An alternative explanation is that the differences observed are a n artifact of the culture system. It is known that culture conditions which DISCUSSION fail to support the ICM of the mouse embryo result in a The progression of preimplantation development in decreased trophoblast proliferation and a n increased the Siberian hamster was slower than that reported in rate of giant cell transformation (see review by Ilgren, the golden hamster (Bavister et al., 1983). Uterine at- 1983). The observed ICM degeneration in the hamster tachment and implantation in the golden hamster be- blastocyst outgrowths may explain the development of gins early on day 4, whereas these events take place extremely large giant cells compared to those of the between 00.00 and 08.00 h r on day 5 in the Siberian mouse. The response of the hamster embryo to differing hamster. The slower rate of development in the Sibe- culture conditions, including the effects of lot-to-lot rian hamster was also evident in later times for com- variation in FCS, must be determined before fundapletion of the third cleavage, cavitation, and hatching mental differences in Siberian hamster and mouse emcompared to the golden hamster. The timing of cleav- bryos can be concluded. Still, i t is apparent that emage and cavitation in the Siberian hamster is similar to bryos from the two species do develop differently under that seen in the mouse, but uterine attachment occurs identical conditions. Further comparative studies of somewhat earlier, since mouse blastocysts can still be implantation in vivo and blastocyst outgrowth in vitro flushed from the uterus on the morning of day 5 (Whit- might provide information not obtainable by the study of the murine rodents alone. tingham, 1971). When blastocysts were recovered early on day 4 and cultured, expansion occurred within a few hours. Full ACKNOWLEDGMENTS expansion of the blastocoel does not appear to take This study was supported by grant HD 25236 from place in vivo before implantation since, without excep- the National Institute of Child Health and Human Detion, both hatched and unhatched blastocysts collected velopment. We thank Dr. Frank Nagy and May Heckon day 4 had small blastocoels and unhatched blasto- man for maintaining the colony of Siberian hamsters. cysts had large perivitelline spaces. This is in contrast to the mouse in which complete expansion of the blasREFERENCES tocoel occurs before hatching in vivo. Blastocyst expanBavister BD, Leibfried ML, Lieberman G (1983): Development of presion is dependent on N a + ,K+-ATPase in the basolatimplantation embryos of the golden hamster in a defined culture era1 surface of the trophoblast pumping N a + into the medium. Biol Reprod 28:235-247. blastocoel (Wiley, 1984). Sodium pump activity in the Iakovenko VV (1974): The estrus cycle of the Djungarian hamster. Arkhiv Anat Gistol Embriol 66:32-36. hamster embryo may be kept low in vivo, possibly due EB (1983): Control of trophoblastic growth. Placenta 4:307to a low N a + / K +ratio in the uterine fluid (Nilsson and Ilgren 328. Ljung, 1985). Incubation in medium with a higher Nieder GL (1990): Protein secretion by the mouse trophoblast during N a + / K + ratio would be expected to stimulate pump attachment and outgrowth in vitro. Biol Reprod 43:251-259. Nilsson BO, Ljung L (1985): X-ray microanalysis of cations (Na, K, activity, resulting in blastocoel expansion.

days of culture. Approximately 50% of hatched blastocysts as well a s some (less than 10%) unhatched blastocysts collected at 21.00 to 23.00 h r on day 4 were able to attach and grow out in our culture system. Figure 3 (a-g) shows a n example of the typical sequence of events during outgrowth. In both the hatched and unhatched blastocysts, trophoblast outgrowth began after a 2 to 3 day lag. Typically, several mobile cells broke off from the attached embryo during the early stage of outgrowth. These cells underwent some cell division before degenerating after 3 to 4 days. The main mass of trophoblast spread as a small number of cells with extremely large nuclei. These giant cells continued to spread for at least another 3 days. The inner cell mass appeared to degenerate during trophoblast outgrowth. Figure 3h shows a mouse embryo cultured for 72 hr, starting on day 4,under identical culture conditions. Hatching was underway within 24 h r of culture, and by 48 h r outgrowth was already extensive. Trophoblast giant cells are more numerous and generally smaller in the mouse than in the Siberian hamster. The small migrating cells typical of the early outgrowth stage in the hamster were not seen in the mouse blastocyst outgrowths.

SIBERIAN HAMSTER EMBRYOS Ca) and anions (S, P, C1) in uterine secretions during blastocyst implantation in the rat. J Exp Zoo1 234:415-421. Pilborough GS (1971): An introduction to the Djungarian hamster (Phodopus sungorus). J Inst Anim Tech 2250-55. Stetson MH, Ray SL, Creyaufmiller N, Horton TH (1989): Maternal transfer of photoperiodic information in Siberian hamsters. 11. The nature of the maternal signal, time of signal transfer, and the effect of the maternal signal on peripubertal reproductive development in the absence of photoperiodic input. Biol Reprod 40:458-465. Weitlauf HM (1988): Biology of implantation. In E. Nobil, J Neil1 (eds):“The Physiology of Reproduction.” New York: Raven Press, pp 231-262. Whittingham DG (1971):Culture of mouse ova. J Reprod Fertil Suppl 14:7-21.

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Wiley LM (1984): Cavitation in the mouse preimplantation embryo: Na/K-ATPase and the origin of nascent blastocoele fluid. Dev Biol 105:330-342. Wimsatt WA (1975): Some comparative aspects of implantation. Biol Reprod 121-40. Wynne-Edwards KE, Terranova PE, Lisk RD (1987): Cyclic Djungarian hamsters, Phodopus campbelli, lack the progesterone surge normally associated with ovulation and behavioral receptivity. Endocrinology 120:1308-1316. Yellon SM, Goldman BD (1984): Photoperiod control of reproductive development in the male Djungarian hamster @‘hodopus sungorus). Endocrinology 114:664-670.

Early embryo development in the Siberian hamster (Phodopus sungorus).

Development of preimplantation embryos of the Siberian hamster (Phodopus sungorus) in vivo and in vitro was examined. The timing of early development ...
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