/. Embryol. exp. Morph. Vol. 41, pp. 65-78, 1977
Printed in Great Britain © Company of Biologists Limited 1977
55
Effects of cyclophosphamide treatment
before implantation on the development of rat
embryos after implantation
By HORST SPIELMANN, 1 HANS-GEORG EIBS
AND HANS-JOACHIM MERKER
From the Departments of Toxicology and Anatomy,
Freie Universitdt Berlin, West-Germany
SUMMARY
After treatment of pregnant rats 24 h before implantation with a single injection of cyclophosphamide (20-80 mg/kg), a dose-dependent increase in resorption was observed at term
but no malformed fetuses could be found. The lowest cyclophosphamide dose that caused
100 % resorption was 60 mg/kg. Somite number and wet weight indicated retardation of
about 24 h during organogenesis. Determination of the time of implantation revealed that the
developmental retardation in treated embryos was not due to delayed implantation. At
implantation, 24 h after cyclophosphamide treatment, a significant and dose-dependent
decrease of the cell number of blastocysts was found. Embryo transplantation experiments
showed that early cyclophosphamide treatment interfered with the subsequent development
of both the embryo and the mother. The decidual reaction seemed to be more affected by the
treatment than the embryos. Most teratologists hold that mouse embryos after treatment in
the preimplantation period either die before implantation or survive to term without being
malformed. The present study, however, proves that the reaction of drugs at this early stage
of pregnancy is more complex than is generally assumed.
INTRODUCTION
The treatment of cleaving rabbit eggs with purine analogues in vivo had no
effect on development until at or after implantation (Adams, Hay & LutwakMann, 1961). In general, however, preimplantation embryos are remarkably
resistant to teratogens; the resulting rarity of birth defects following treatment
during this period has been explained as follows (Austin, 1973): 'The effect of
teratogens on the cleavage embryo depends on the number of cells killed or
inhibited: above a certain portion, the embryo dies; below that figure, the
remaining cells multiply to replace those lost and subsequent development is
essentially normal'. This view is still shared by most teratologists (e.g. Wilson,
1973).
A different approach to this problem has been used by Gottschewski and
1
Author's address: Institut fur Toxikologie und Embryonal-Pharmakologie der Freien
Universitat Berlin, Garystr. 9, D-1000 Berlin 33, West-Germany.
66
H. SPIELMANN AND OTHERS
coworkers (Gottschewski, 1963, 1964; Gottschewski & Zimmermann, 1963).
After treatment during the preimplantation period they inspected rabbit
embryos during organogenesis. When cyclophosphamide (CPA) (40 mg/kg
intravenously) was given to pregnant rabbits before implantation, a high percentage of deformed fetuses was found on days 11, 17 and 30 p. c. (post-coitum),
suggesting that the substance may penetrate into the blastocysts even before
implantation and interfere with normal development.
Gottschewski's studies were later extended to the rat (Brock & von Kreybig,
1964). When CPA was given on day 3 p.c. and the uteri were examined on day
12, the number of resorbed and malformed embryos was significantly increased.
On day 15 p.c. all malformed embryos were resorbed so that only resorbed or
completely normal embryos could be seen.
Mice injected with CPA on day 3 p.c. showed an increased resorption rate,
but no malformed embryos at the end of gestation (Gebhardt, 1970). Administration of CPA to pregnant rabbits at about the time of implantation (day 6
and 7) led to an increase in the number of foetal deaths (Fritz & Hess 1971)
and about 10 % of the foetuses from dams treated on day 7 exhibited malformations at term.
To get further information on the mechanism of action of teratogens given
during the preimplantation period we repeated and extended earlier studies
(Brock & von Kreybig, 1964) with CPA on the rat. We tried to determine the
time of death of such embryos and whether embryos that die are malformed or
only retarded in development. We also attempted to study the development of
treated embryos before and around the time of implantation. Finally we used
the embryo transfer technique to see whether CPA during the preimplantation
period predominantly affects the embryo or the mother.
MATERIALS AND METHODS
Wistar rats of the strain SW 72 weighing 200 g (breeder Winkelmann, Kirchborchen, Germany) were kept under a normal day/night cycle and placed with
males overnight. The presence of spermatozoa in vaginal smears indicated day 0
of pregnancy. Implantation occurs in this strain between 120 and 132 h after
the midpoint of the overnight mating period, this is between day 4 and day 5.
Blastocysts were usually obtained at 2 p.m. on day 4, 24 h after the time of
treatment. CPA, which was a gift from Prof. N. Brock (Asta-Werke AG,
Chemical Factory Brackwede, Germany), was dissolved in distilled water and
injected subcutaneously.
In the transplantation experiments and in the course of the determination of
cell number, embryos were handled in Whitten's medium (Whitten, 1971),
freshly prepared from three-times quartz-distilled water. The cell number of the
preimplantation embryos on day 4 was determined by the method of Tarkowski
(1966) and the mitotic index (number of cells in mitosis as percentage of total
Cyclophosphamide action on early embryos
61
cells) was calculated from the same preparations. To follow the decidual reaction, 0-5 ml of a 1 % Pontamine sky blue solution was injected into a tail vein
(Finn & Martin, 1972).
In the embryo transfer experiments blastocysts were surgically transplanted
in groups of five to one uterine horn of pseudopregnant females on day 4 of
pseudopregnancy. The pseudopregnant recipients were anaesthesized with
Evipan (hexobarbital) which was a gift from the Bayer AG (Pharmaceutical
Company, Germany). Pseudopregnancy was induced by mating normal females
with vasectomjzed males. The foster mothers were killed on day 20 of pregnancy
and the success rate of the transplantations was determined by the number of
resorbed and live embryos. The embryos were weighed, inspected for growth
retardation and malformations, and stained for skeletal abnormalities with
Alizarin red S (Lorke, 1965).
In the morphological studies the treated mothers were killed at different times
during pregnancy, the uteri were excised and the embryos were removed from
the implantation sites on days 11, 14, 17 and 20. The embryos were checked for
developmental anomalies under a stereo microscope (Carl Zeiss, Oberkochen,
Germany) and their somite numbers determined.
For histological examination uterine horns of pregnant mice of day 11 or 14
of gestation were excised, fixed with embryo undisturbed in Carnoy's solution,
and embedded in paraffin. Serial sections in two directions (cross and longitudinal) were prepared. Sections were routinely stained with haematoxylin and
Eosin, PAS, or Azan. Only material which clearly showed an implantation site
without too heavy haemorrhage was chosen for histological studies.
RESULTS
1. Lethality rate at term after CPA treatment on day 3
As described by Brock & von Kreybig (1964), groups of ten pregnant rats
received a single s.c. injection of 20, 40, 60 or 80 mg/kg CPA on day 3. The
maternal LD 50 in our strain is 180 mg/kg. The resorption rates at term for the
four doses were 48 % 82 %, 100 % and 100 %, compared with 10 % for control
SW 72 embryos (see Fig. 1).
The wet weight of the living embryos at term was not reduced in the four
treated groups, and no malformations were observed.
2. Changes in the number of dead and living fetuses after CPA treatment
(60 mgjkg) on day 3
Pregnant animals were treated with one injection of 60 mg/kg CPA on day 3
and the number of embryos recorded at different times after treatment. After
24-48 h, i.e. on days 4 and 5, the total number of embryos was calculated from
the number of implantation sites visible after Pontamine sky blue injection and
the number of embryos (blastocysts) that could be flushed from the same
68
H. SPIELMANN AND OTHERS
Dead
Control embryos
| Living
HI Dead Treated embryos
H Living (day 3, 60 mg/kg
Cyclophosphamide)
3
4*5
Implantation
11
14
Day of pregnancy
20
Fig. 1. Influence of cyclophosphamide treatment 24 h before implantation on the
number of embryos/female in the rat. Columns represent means from ten animals
per day and treatment group. Details for the time of implantation (between days 4
and 5) are given in Table 1.
uterus. On days 8, 11, 14 and 20 the number of implantation sites could easily
be determined. On days 11, 14 and 20 all implantation sites were dissected
under the stereo microscope and inspected for living or dead embryos. Ten
treated and ten control animals were investigated on each day.
The results are given in Fig. 1. There are no significant effects of CPA treatment on the total number of implantations per female. In both groups there is
a reduction of the number of embryos at the time of implantation. The most
striking reduction in the number of living embryos in the treated group occurred
during organogenesis, as indicated by the decrease from 70 % to 10 % between
days 11 and 14.
3. Development of rat embryos during organogenesis (day 11 and 14) after
maternal treatment with 60 mg/kg CPA on day 3
We tried to confirm earlier observations (Brock & von Kreybig, 1964) on
malformations of rat embryos during organogenesis caused by CPA treatment
during the preimplantation period. Among 30 litters observed under the stereo
microscope we never found any malformations of the brain or heart as described
previously (Brock & von Kreybig, 1964). The embryos seemed to be retarded
in development rather than grossly malformed (Spielmann, 1976).
The somite number per embryo is significantly lower in treated embryos
(Fig. 2). According to the developmental stage given by the somite number per
Cyclophosphamide action on early embryos
69
60 i-
50
40
| 30
20
10
12
13
Day of pregnancy
14
Fig. 2. Influence of cyclophosphamide treatment on day 3 of gestation on the somite
number of embryos during organogenesis. Values are given ± standard deviation,
the numbers indicate the amount of embryos used in each determination. • — # ,
Control embryos; *—*, treatment on day 3 with 60 mg/kg cyclophosphamide.
embryo in Fig. 2, the treated embryos seem to be retarded by about 24 h. Dry
weight determinations of treated and untreated embryos gave the same results.
In contrast to previous investigators (Brock & von Kreybig, 1964), we conclude
that rat embryos treated with CPA in the preimplantation period die during
organogenesis without being malformed and that these embryos are retarded
in development by about 24 h. Histological inspection of control and treated
uteri during organogenesis revealed a disturbed development of the placenta,
e.g. on day 14 (Fig. 3).
70
H. SPIELMANN AND OTHERS
id)
Fig. 3. For legend see facing page.
Cyclophosphamide action on early embryos
71
Table 1. Number of embryos per female on days 4-5 after
treatment with 60 mg/kg cyclophosphamide on day 3*
Time of
determination
Day 4
4-5
5
,
Control embryos
*
>
Not implanted
Implanted
7
4
2
7
10
10
Treated embryos
A
,
*
Not implanted
Implanted
5
2
1
8
9
11
* Based on means from 10 animals for each time of determination.
4. Development of rat embryos at the time of implantation {day 4 and 5) after
maternal treatment with 60 mgjkg CPA on day 3
The developmental retardation of 24 h found in treated embryos during
organogenesis could be explained by delayed implantation. To determine the
exact time of implantation for treated and untreated animals, the implanted
and unimplanted embryos were counted in both groups on day 4, day 4-5
(12 h later) and day 5. Table 1 shows that there were only slight differences in
the time of implantation between the treated and untreated groups. There is no
indication of a delayed implantation in the treated group.
5. Effects of CPA treatment on pregnant rats on day 3 of embryonic development
before implantation
To find out whether the abnormal embryonic development of rat embryos
during organogenesis after maternal treatment on day 3 with 60 mg/kg CPA is
caused by an action of the drug predominantly on the mother (decidual reaction)
or on the embryo or on both, we studied the development of treated embryos
before implantation. Groups of ten pregnant females were treated with single
FIGURE 3
Light microscopy of control uteri and treated uteri on day 14 of gestation.
(a, b) Controls (Fig. 3 a = xJ5 and Fig. 3 b = x 45). The amniotic cavity (A) is occupying more than $ of the space inside the uterine wall. The sickle-shaped placenta
(P) and decidua are only filling a small zone. The layers of the uterine wall - placenta
(P), decidua (D), smooth muscles (M) - can easily be distinguished,
(c, d) Treatment of the female on day 3 with 60 mg/kg CPA (Fig. 3 c = x 15 and Fig.
3d = x 50). Both diameter and thickness of the placenta are considerably smaller
than in controls. Histological inspection is hampered, since the layers of placenta and
decidua are not running straight but are indented and vary in thickness. Prominent
features are the expansion of the trophoblast giant cell layer (T) and the reduced
thickness of the placenta (P). In some areas the decidua (D) has not been pushed to
the periphery during placental development. In such regions the placenta is covering
the decidua islands and is being protruded into the amniotic cavity. There are still
remnants of the uterine cavity.
72
H. SPIELMANN AND OTHERS
Table 2. Cell number of rat embryos at implantation {day 4)
24 hours after cyclophosphamide treatment
Time of
determination
Dose
(mg/kg)
Day 3 (controls)
4 (controls)
4 (treated)
4 (treated)
4 (treated)
Cell number
per embryo
Number of
determinations
Mitotic index
(%)
47
128
72
78
102
50
40
3-8
4-5
5-3
14-6±4-2
27-7±4-5
19-9 ±39*
17-3 + 4-3*
14-2 ±5-8*
20
40
60
Mitotic index: Cells in mitosis expressed as percentage of total cells of the embryos.
* = significantly lower than cell number of controls on day 4 at P < 0-001.
injections (subcutaneously) of 20, 40 or 60 mg/kg. CPA on day 3 and the uteri
were flushed 24 h later, on day 4. The number of blastocysts per female was not
reduced in any of the treated groups.
However, the cell numbers of the treated blastocysts (see Table 2) were
significantly lower than those of control blastocysts (P < 0-001), and the cell
numbers of the blastocysts in the 60 mg/kg group were significantly lower than
those of the 20 mg/kg group (P < 0-001). The cell number as well as the mitotic
index of the blastocysts treated with the highest CPA concentration (60 mg/kg)
is the same as that of normally developing control morulae of the same strain
(see Table 2). The considerable reduction of the cell number of the treated
blastocysts indicates that CPA or one of its metabolites interfered with embryonic development during the 24 h of preimplantation development between
day 3 and day 4.
Table 3. Influence of maternal cyclophosphamide treatment {day 3; 60 mgjkg)
on development of rat blastocysts after transplantation to pseudopregnant recipients on day 4*
Untreated
control
(A) Number of
embryos transferred
(B) Implantations
(percentage of A)
(Q Living embryos
(percentage of A)
(D) Resorptions
(percentage of A)
Blastocyst treated
recipient untreated
Blastocyst untreated
recipient treated
85
115
115
72(85%)
103 (90 %)
75 (65 %)
40(47%)
14(12%)
14(12%)
32(38%)
89 (78 %)
61 (53 %)
Five blastocysts transferred to one uterine horn ol a recipient which was killed on day 2U
for determination of success rate.
Fig. 4. Light microscopy (magnification approximately x 15) of the transplantation
experiment during organogenesis (day 11).
(a) Control, day 11 (x 12). On day 11 the normal decidua (D) fills half of the space
internal to the unstriated muscles of the uterine wall. The amniotic cavity (A)
with the relatively small embryo is occupying the other half. The uterine lumen is
almost completely compressed or obliterated in the periphery of the decidua. P =
placenta.
(b) Transplantation control, day 77 (x 12). After transplantation of blastocysts to
pseudopregnant hosts on day 4, the light microscopic picture of most of the embryos
on day 11 corresponds to the morphological situation of the control group. The
decidua (D) of some animals, however, seems to be slightly smaller. Moreover, part
of the uterine lumen (L) can still be demonstrated.
(c) Transplantation of a treated embryo (60 nig/kg CPA on day 3) to an untreated
pseudopregnant recipient, day 11 (x 17). Under these conditions the decidua (D) is
normally developed. However, the placenta anlage (P) and the embryonic-maternal
transitional region are smaller than in control animals. In some instances the
amniotic cavity (A) is smaller and the deciduafillstwo thirds of the area in the central
section. The decidua capsularis (X), which is normally compressed to a narrow
band, is still comparatively wide.
(c) Transplantation of an untreated embryo to a treated pseudopregnant recipient
(60 mg/kg CPA on day 3), day 11 (x 17). When the recipient is treated 24 h before
transplantation, the decidua (D) is considerably smaller. The size of the placentae
of most of the embryos is clearly diminished. The uterine lumen (L) is in all cases
widely open. It is usually filled with blood and fibrin. Here and there cells of maternal and embryonic origin penetrate into these cavities. Fibrous filaments and
numerous trophoblast giant cells can still be observed.
74
H. SPIELMANN AND OTHERS
id)
Fig. 5, For legend see facing page.
Cyclophosphamide action on early embryos
75
6. Transplantation experiments after maternal treatment with CPA during the
preimplantation period
Following treatment during the preimplantation period, the transplantation
of preimplantation embryos distinguishes between effects of an agent on the
mother and direct effects of the agent on the embryo (Finn & Bredl, 1973;
Spielmann, 1976). We therefore transplanted blastocysts on day 4 (24 h after
maternal treatment) to pseudopregnant recipients on day 4 of pregnancy. We
also transplanted untreated blastocysts on day 4 to pseudopregnant recipients
on the same day of pregnancy which had been treated with CPA on day 3 (24 h
before transplantation). Untreated blastocysts transplanted on day 4 to untreated pseudopregnant recipients on day 4 of pregnancy served as controls.
Table 3 shows that in untreated control experiments 47 % of the blastocysts
developed into viable fetuses at term. The percentage of living fetuses at term
was reduced to the same extent in both treated groups (12 %). No indication of
malformations could be found among the living term fetuses from the control
FIGURE 5
Light microscopy (approximately x 50) of placenta and decidua of the transplantation experiment during organogenesis (day 1.1).
(a) Control, day 11 (x 50). Under the muscle layer a zone of small, loosely packed
connective tissue cells with distinct intercellular spaces can be observed (X). Towards the centre, i.e. towards the placental anlage, the cells are getting bigger, the
intercellular spaces disappear, and the tissue becomes epithelium-like (D). The
vessels radiate concentrically towards the placental anlage. At this point of development the disc-shaped placenta (P) already contains numerous blood-filled lacunae
and projects slightly into the amniotic cavity. The transitional field between placenta
and decidua reaches pyramidally into the maternal tissue and contains vessels,
decidual cells, and giant cells of the trophoblast (T).
(b) Transplantation control, day 11 (x50). The morphological picture corresponds
to the control group. The number of trophoblast giant cells (T) in the embryonicmaternal transitional region is increased. Indices identical to 5 a.
(c) Transplantation of a treated embryo (60 nig/kg CPA on day 3) to an untreated
pseudopregnant recipient, day 11 (x 50). The decidua is normally developed but at
the same time the placental anlage and the embryonic-maternal transitional region
are smaller than in controls. The small amount and size of blood-filled lacunae are
conspicuous. The number of trophoblasts giant cells is smaller or in some cases even
increased. Indices identical to 5a.
(d) Transplantation of an untreated embryo to a treated pseudopregnant recipient (60
mg/kg CPA on day 3), day 11 (x 50). The size of the placentae of most of the embryos
is clearly diminished and the decidua is considerably smaller than in controls. The
peripheral zone of the decidua with loosely packed cells is wider. An epitheloid
packing of cells is only to be seen in a small central area. These central cells often
contain large glycogen deposits and fatty inclusions. The number of trophoblast
giant cells is moderately increased. In a few instances the placental anlage consists
only of a band of trophoblast giant cells (T); the pyramidal embryonic-maternal
transitional field is usually absent in these uteri. Lacunae can rarely be found. A
few embryos have a typically developed placenta which is always smaller than in
untreated day 11 uteri. Indices identical to (a).
76
H. SPIELMANN AND OTHERS
transplantations nor from the transplantations with pretreated embryos or
recipients. We therefore conclude that CPA given to rats on day 3 of gestation
interferes with the development of the embryo before implantation and also
with the decidual reaction of the uterus.
7. Histological examinations of the transplantation experiments during organogenesis (day 11, Fig. 4 and Fig. 5)
The uterine histology of untreated transplantation controls (Figs. 4b and 56)
did not differ from that of normal controls on the same day of pregnancy
(Figs. 4a and 5a). When a treated embryo is transplanted to an untreated foster
mother (Figs. 4 c and 5 c), the embryo-derived placental anlage and the embryomaternal transitional region are smaller than in controls. Finally, when an
untreated embryo is transferred to a pretreated recipient (Figs. 4 d and 5d),
the maternal part of the implantation site, the decidua, is considerably smaller
than in controls. This again suggests an action of the CPA treatment on both
the embryo (Figs. 4 c and 5 c) and the uterus.
DISCUSSION
The present study clearly demonstrates that full information on the effects of
different agents on preimplantation embryos cannot be obtained when evaluation of the resulting developmental abnormalities is performed at term only,
since the damaged embryos rarely survive up to birth. We confirmed the finding
of Brock and von Kreybig (1964) that after CPA treatment of pregnant rats
during the preimplantation period the embryos die during organogenesis. In
contrast to the earlier findings, treated embryos were not malformed and were
retarded in development by about 24 h, as judged by the somite number and
the dry weight of the embryos. After our treatment, even though it is performed
before implantation, the embryos neither die during implantation nor survive
to term, but survive up to organogenesis. The common view on drug action
before implantation has, therefore, to be modified.
CPA was shown to have an effect on embryonic development prior to implantation, as revealed by the reduced cell number of the treated blastocysts.
There are several reports on embryos that degenerated after in vivo treatment
during the preimplantation period, e.g. when using X-rays (Russell, 1950) or a
zinc-deficient diet (Hurley & Shrader, 1975). However, this is to our knowledge
the first report on preimplantation embryos that appeared morphologically
normal after maternal drug treatment but had a reduced cell number.
The embryo transplantations, and also the histological examination of the
uteri of the pretreated recipients, demonstrated an effect of the alkylating drug
on the decidual reaction of the uterus. The survival rate of pretreated blastocysts
was also reduced at term. These results provide additional evidence for a direct
effect of the drug or one of its metabolites on the embryo. On day 11 the histo-
Cyclophosphamide action on early embryos
11
logical examination of the uteri of untreated hosts to which treated embryos
had been transplanted point in the same direction, since the placentae show signs
of retarded or abnormal development but the decidua is not affected. The present data give no indication whether the retarded development of the embryos
is due to an action of the drug on the embryo or to secondary effects caused by
the inhibited development of the decidua. The effects of the early treatment
on later stages of pregnancy might be explained by a retarded clearance of the
teratogenic agent from the blastocoele.
Investigations on the teratogenic effect of CPA during organogenesis indicate
that the unmetabolized compound seems to be teratogenic (Gibson & Becker,
1968, 1971). This is in contrast to the alkylating activity of the drug, which is due
to metabolites (Brock, 1967; Sladek, 1973).
Chemically induced chromosome aberrations in the mouse produce embryonic mortality in the pre- as well as in the postimplantation period (Basler,
Buselmaier & Rohrborn, 1976). Malformations could not be found in these
embryos during organogenesis, though chromosome anomalies were easily
detectable. Since CPA is a very potent mutagenic agent (Rohrborn & Buckel,
1976), the abnormal development of our treated embryos could be caused by
somatic mutations and subsequent chromosomal imbalance.
This work was supported by grants of the Deutsche Forschungsgemeinschaft awarded
to the Sfb 29-Embryonale Entwicklung und Differenzierung. We thank Dagmar Nagel,
Ursula Jakob, Imke Dillmann and Helga Stiirje for their technical assistance, Barbara Steyn
for her editorial assistance and Thomas Kwasigroch for his comments on the manuscript.
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