/ . Embryol. exp. Morph. Vol. 32, 3, pp. 739-748, 1974
739
Printed in Great Britain
Trophoblastic factors involved in lysis
of the blastocyst coverings and in implantation
in the rabbit: observations on inversely
orientated blastocysts
By H.-W. DENKER 1
From the Arbeitsgruppe Prof. Dr G. H. M. Gottschewski, MaxPlanck-Institut fur Immunbiologie, Freiburg {Germany)
SUMMARY
Lysis of the blastocyst coverings in the rabbit normally starts at the abembryonic hemisphere
of the blastocyst facing the antimesometrial endometrium. Theoretically, this restricted
effect could be caused by factors locally produced by abembryonic trophoblast, antimesometrial endometrium, or a combination of them. Blastocysts abnormally implanting
with reversed orientation exhibit lysis of blastocyst coverings in the abembryonic hemisphere
despite the absence of antimesometrial endometrium, and they do not show lysis in the
embryonic hemisphere despite its contact with antimesometrial endometrium. Accordingly,
it appears that lysis of blastocyst coverings is attributable to a factor localized in the
abembryonic hemisphere of the blastocyst. Blastocyst protease activity was found to be
maximal there in all blastocysts, whether oriented normally or reversed. The origin and
role of this enzyme and other factors are discussed.
INTRODUCTION
Controversial views have been presented about the respective role of trophoblast, blastocyst coverings (zona pellucida), uterine tissues and uterine secretion
in implantation of the mammalian egg. Discussions of these different aspects
can be found in literature as early as the very beginning of this century (Graf
von Spee, 1901; Grafenberg, 1910; Halban & Frankl, 1910; Caffier, 1929).
Recently, preimplantation rise in uterine secretion protease activity has been
demonstrated in the mouse (Mintz, 1971, 1972) and in the rabbit (Schwick,
1965; Denker, 1969; Kirchner, Hirschhauser & Kionke, 1971; Kirchner, 1972).
Evidence has been shown that a uterine protease might, in the mouse, cause
the lysis of the zona pellucida and act as an implantation-initiating factor
('IIF') (Mintz, 1971, 1972). According to this concept, the process depends
mainly on uterine factors (protease), the trophoblast playing a passive role.
The same point of view has been presented for the rabbit (Kirchner et ah
1
Author's address: Arbeitsgruppe Prof. Dr G. H. M. Gottschewski am Max-PlanckInstitut fur Immunbiologie, 78 Freiburg, Stefan-Meier-StraBe 8, Germany (BRD).
740
H.-W. DENKER
1971; Kirchner, 1972, 1973), although this species exhibits a different type of
implantation (the central type), and the zona pellucida forms only a minor
part of its blastocyst coverings (Boving, 1957; Denker, 1970 b). On the other
hand, protease activity has been demonstrated in the blastocyst of the guinea
pig (Blandau, 1949; Owers, 1970), rabbit (Denker, 1969; Kirchner, 1972), and
mouse (Andary, Dabich & Van Winkle, 1972). In the rabbit, substrate film
tests demonstrate higher levels of protease activity associated with the blastocyst than found in the uterine secretion. The blastocyst enzyme was initially
thought to be secreted by the trophoblast (Denker, 1969). Later on the main
activity could be localized in the blastocyst coverings (Kirchner, 1972; Denker,
1974). Since the enzyme molecule cannot be synthesized at this extracellular
site we have to consider whether it is a product of the maternal genital tract
or of the trophoblast. Kirchner (1972) favours the view that it originates from
the maternal genital tract secretions, and that activation might take place at
the surface of the blastocyst.
Lysis of blastocyst coverings starts, in the rabbit, normally at about 7 days
p.c. {post coitum) when the blastocyst is already orientated in the uterus with
its embryonic disc towards the mesometrium. The coverings first disappear
in the abembryonic-Iateral region of the trophoblast facing the antimesometrial
endometrium (cf. Discussion). As long as only normally orientated eggs are
being studied, it is impossible to recognize whether the lysis-initiating factors
are produced by the endometrium or by the apposed trophoblast.
We had the chance of studying, among several hundreds of rabbit blastocysts sectioned during the last years, three cases of inverted orientation at or
after 7 days p.c. These observations will be described in the present communication. Attention is focused on the lysis of the blastocyst coverings and
on related protease activity. So far, no way has been found of producing
abnormal orientation of the blastocyst experimentally. Therefore observations
like these can be made only by chance, and the number of observations necessarily remains small.
MATERIALS AND METHODS
Female rabbits were individually caged and fed a standard pellet diet and
water ad libitum. Each was mated with two fertile bucks. Further details about
the animals are given in the section on individual observations. After sacrifice
(animals 1 and 3 by stunning and bleeding, animal 2 by intra-venal injection
of an overdose of sodium pentobarbital), uteri were quickly removed and
quenched with liquid nitrogen. Unfixed sections were cut on a cryostat at
- 2 5 °C and at a thickness of 14 /im. For morphological observations they
were mounted on slides, air-dried and stained with toluidine blue in 30 %
ethanol after Kramer & Windrum (Pearse, 1968). Protease activity was determined using the improved gelatin substrate film technique (Denker, 1974).
Lysis of coverings in inversely orientated blastocysts
741
RESULTS
For comparison, features of a normally orientated blastocyst are pictured
in Fig. 1A-C. The embryonic disc is located mesometrically, the trophoblast
with trophoblastic knobs faces the antimesometrial and lateral endometrium;
the blastocyst coverings have disappeared in the abembryonic/antimesometrial
region, but they are retained over the embryonic disc (mesometrially). The
protease activity is maximal in the abembryonic/antimesometrial area. At the
embryonic pole (mesometrially), activity is moderate at 7 days p.c. and very
low at 1\ days p.c. (Fig. 2 A).
Animal 1 (12L). 7 days 8h/?.c. Mixed-bred rabbit, 8^ months old. Primigravida. Number of corpora lutea: R (right) 2, L (left) 8; number of blastocysts:
R2, L7. All blastocysts were found to be evenly spaced and of regular size.
The abnormally orientated blastocyst was found in the left uterus. The
apparently normal embryonic disc could be identified clearly at the antimesometrial side of the uterus (Fig. IE). The abembryonic trophoblast was
facing the mesometrial endometrium including the placental folds (Fig. 1F).
Here normally appearing trophoblastic knobs were present, but none of them
was found to contact or to invade the uterine epithelium. The blastocyst
coverings were well preserved in the region of the embryonic disc (antimesometrially, Fig. 1D), but they had disappeared from the abembryonic, trophoblastic hemisphere (mesometrially, Fig. IF). Protease activity (as indicated by
the width of the lysis zone in the substrate film) was highest abembryonally
(mesometrially); the blastocyst coverings at the embryonic pole exhibited
moderate activity. An inflammatory infiltration and exudate (mostly lymphocytic) were found in a localized area of the antimesometrial endometrium
near the blastocyst.
Animal 2 (Dtl3). 7 days p.c. New Zealand White rabbit, about one year
old. This was an experimental animal which had received, during laparotomy
performed under sodium pentobarbital anaesthesia at 15 h p.c, Sephadex
beads transferred to the oviducts (for further details see Denker & Hafez,
1974). Number of corpora lutea: R9, L5; number of blastocysts: R7, L4. All
blastocysts were normally spaced and of regular size.
One of the blastocysts found in the right uterus was abnormally orientated:
the trophoblast was directed toward the mesometrial side of the uterus.
Unfortunately no detailed observations could be made on the embryonic disc
which was not satisfactorily preserved in the sections. The blastocyst coverings
were present in the antimesometrial hemisphere but absent mesometrially.
Some protease activity was found all around the blastocyst (the typical pattern
of the 7 days p.c. stage), but lysis zones were wider in the mesometrial than
in the antimesometrial region.
Animal 3 (12A). 8 days p.c. Mixed bred rabbit, 12 months old, weight
3600 g. Primigravida. This was an experimental animal ovariectomized at
742
H.-W. DENKER
Lysis of coverings in inversely orientated blastocysts
743
6 days/?.r. and injected with progesterone (for further details see Denker, 1972).
Number of corpora lutea: R6, L6; number of blastocysts: R6, L6. All blastocysts were evenly spaced; all of them were of regular size except two in the
left uterus, which were small.
One of the regular size blastocysts of the left uterus was abnormally orientated : the well developed embryonic disc faced the antimesometrial endometrium.
The trophoblast was orientated mesometrially; some of the trophoblastic knobs
appeared to have penetrated the mesometrial uterine epithelium, but this
detail could not be observed clearly in the cryostat sections. Remnants of the
blastocyst coverings were still present and well preserved in the region of the
embryonic disc (antimesometrially). More swollen and crumpled remnants
could be seen in the paraplacental furrow (mesometrial part of the uterus)
whereas the trophoblast itself was uncovered. Protease activity was low and
present only in a few scattered areas, mostly between trophoblast and antimesometrial endometrium.
DISCUSSION
In the rabbit, the blastocyst coverings do not disappear all around the egg
at the same time: lysis starts first at the abembryonic-lateral part of the blastocyst which, in normally orientated eggs, faces the antimesometrial part of the
endometrium (cf. Boving, 1963, p. 333, and fig. 18, p. 353; Denker, 1970a,
Fig. 1. Morphology. 7£ days post coitum (p.c). Cryostat sections, toluidineblue stain.
A-Fx9O;GxlO.
A-C, Normally orientated blastocysts.
A, Embryonic disc (below) facing the mesometrial endometrium (above). The
intensely stained line represents the blastocyst coverings.
B, The same region as shown in A but from another blastocyst; in this section
the embryonic disc is morphologically better preserved but the blastocyst coverings
show less stain uptake than in A.
C, Abembryonic trophoblast (above) apposed to the antimesometrial endometrium (below). Two trophoblastic knobs can be seen. There are no remnants of
the blastocyst coverings in this region.
D-G, Inversely orientated blastocyst (12L).
D, Embryonic disc (above) facing the antimesometrial endometrium (below).
The intensely stained line represents the blastocyst coverings.
E, Another section of the same area as shown in D; toluidine blue staining
intensity of the blastocyst coverings varies especially after dehydration in alcohol,
so in this specific section the coverings are, in contrast to D, unstained and appear
as a white band between embryonic disc and endometrium.
F, Abembryonic trophoblast (below) apposed to the mesometrial endometrium
(above). Two trophoblastic knobs can be distinguished. The blastocyst coverings
are lysed in this region.
G, Low magnification. The intensely stained line on top of the antimesometrial
endometrium (below) represents the blastocyst coverings.
744
H.-W. DENKER
Lysis of coverings in inversely orientated blastocysts
745
fig. 46, p. 212). At the embryonic pole (mesometrially), the coverings remain
intact and easily demonstrable for some time. The controversial description
given by Kirchner (1972) can hardly be sustained when appropriately stained
sections are studied (cf. Fig. 1A-C). In the ferret basically the same pattern
has been found: the blastocyst coverings are lysed first in the abembryonic
hemisphere, while they are still retained in the region of the embryonic disc
(which unlike that in the rabbit is located antimesometrially in this species)
(Enders & Schlafke, 1972).
Concerning the mechanisms causing the local onset of lysis of the blastocyst
coverings in the abembryonic (and, in the rabbit, antimesometrial) region, we
are discussing three possibilities:
(1) Lysis might be caused by uterine proteases (or other enzymes) present
in the uterine secretion all around the blastocyst, but the sensitivity of the
coverings might be different at the embryonic and the abembryonic pole. So
far no evidence has been found to support this hypothesis, in fact the composition of rabbit blastocyst coverings seemed to be the same all around the
circumference of preimplantation blastocysts (Denker, 1970 a,b).
(2) One or more factors necessary for lysis (e.g. proteases, other enzymes,
activators) might occur antimesometrially exclusively or in greater abundance
than elsewhere. The target then would primarily be that part of the blastocyst
which faces this antimesometrial portion of the endometrium. Provided normal
orientation has taken place, this is, in the rabbit, the abembryonic part of the
blastocyst. But if the blastocyst would happen to become trapped with the
embryonic pole towards the antimesometrial endometrium, lysis could be
expected to start at this atypical part of the blastocyst (Fig. 3B).
(3) The site of production of the above mentioned factors (see (2)) is the
abembryonic trophoblast. These factors would then be acting on the abembryonic part of the coverings, in normal and also in case of abnormal
orientation of the blastocyst in the uterus (Fig. 3 A, C).
In all three cases of inverted orientation described in the present paper,
lysis of the blastocyst coverings had occurred in the 'proper' (abembryonic,
Fig. 2. Protease activity. 7 | days p.c. Cryostat sections, gelatin substrate film test.
A, B, x9; C, D, x90.
A, Normally orientated blastocyst, embryonic disc mesometrially. Protease activity
as indicated by the unstained lysis zone is found nearly exclusively in the antimesometrial (abembryonic) region (below).
B, Inversely orientated blastocyst (12L), embryonic disc located antimesometrially.
Main protease activity found in the mesometrial (abembryonic) region (above).
C, D, The same blastocyst as in Fig. 2B, another section, higher magnification.
C, Mesometrial region. High activity in the space between trophoblast and
endometrium.
D, Antimesometrial region. The blastocyst coverings (located between embryonic
disc, above, and endometrium, below) only locally exhibit some activity.
746
H.-W. DENKER
Normal orientation
Inverse orientation
(Found)
(Not found)
Endometrial factor
Trophoblastic factor
Fig. 3. Diagrams to show possible factors involved in the lysis of the blastocyst
coverings in A, normal orientation, and B and C, inverse orientation. In the case
of normal orientation of the blastocyst in the uterus (embryonic disc towards the
mesometrium, A), it is impossible to decide whether it is factors provided by the
abembryonic trophoblast, or factors derived from the antimesometrial endometrium
that trigger the onset of lysis of the blastocyst coverings. In the case of inverse
orientation (embryonic disc located antimesometrially, B and C), antimesometrial
onset of lysis (here the embryonic disc region, B) would indicate that it depends
on specific endometrial factors located antimesometrially, whereas lysis in the
mesometrial region (here the abembryonic trophoblast region, C) would indicate
that it is started by factors provided by the abembryonic trophoblast.
trophoblastic) hemisphere of the blastocyst, although this hemisphere faced
the 'wrong' part of the endometrium: the mesometrial endometrium (Fig. 3C).
At the embryonic pole, located antimesometrially, remnants of the coverings
were still present, even as late as 8 days p.c. Evidently the lysis initiating agent
is not secreted locally by the antimesometrial endometrium but rather by the
Lysis of coverings in inversely orientated blastocysts
trophoblast. This view is sustained by observations that the lysis starts from
the trophoblastic knobs which erode the coverings from inside (Boving, 1963,
fig. 17, p. 352; Enders & Schlafke, 1969, p. 4; Denker, 1970a, fig. 40, p. 203;
Steer, 1970, fig. 3, p. 318).
The three cases of abnormal orientation described above occurred under
abnormal conditions, as described under 'observations' (treatment of the
animal, inflammation). Interpretation of the physiological implications of the
described features has to take this into account and has to be done cautiously.
On the other hand there is a remarkable regularity in the observations made:
the lysis of the blastocyst coverings started, in all three cases, at the 'right'
pole of the blastocyst, and at the 'wrong' side of the uterus, giving evidence
for local trophoblastic factors but not for local endometrial factors. This view
is further substantiated by the observation that egg coverings do not gain
normal protease activity in the uterus if no blastocyst tissue is present (Denker
& Hafez, 1974). In the case of our inversely orientated blastocysts, maximum
protease activity was, in animals 1 and 2, found in the region where the lysis
of the coverings was most advanced, and this was again the region of the
abembryonic trophoblast, and it did not seem to be determined by the special
region of the apposed endometrium. Animal 3 was too late a stage for the
study of the protease; at 8 days p.c. only residual activity is found in the
normal situation (Denker, 1969).
The present communication presents evidence that one or more trophoblastic factors are essential for the lysis of the noncellular coverings of the
rabbit blastocyst. The identification and origin of the protease, other enzymes,
activators and inhibitors, uterine as well as blastocystic, are objectives of
continuing investigation.
The author wishes to express his gratitude to Dr Bent G. Boving (Wayne State University
School of Medicine, Detroit, Michigan, USA) for his discussions and valuable suggestions,
and to Dr E. S. E. Hafez for giving him the opportunity to do part of this work during
a sabbatical leave at the C. S. Mott Center for Human Growth and Development, Wayne
State University School of Medicine, Detroit, Michigan, U.S.A. This investigation was
supported by the Deutsche Forschungsgemeinschaft (Grant De 181/3).
REFERENCES
T. J., DABICH, D. & VAN WINKLE, L. J. (1972). Changes in proteinase activity in
early vs. late mouse blastocysts. /. Cell Biol. 55, 3 a.
BLANDAU, R. J. (1949). Embryo-endometrial relationships in the rat and guinea pig. Anat.
Rec. 104, 331-359.
BOVING, B. G. (1957). Rabbit egg coverings. Anat. Rec. 127, 270.
BOVING, B. G. (1963). Implantation mechanisms. In Conference on Physiological Mechanisms
Concerned with Conception (ed. C. G. Hartman), pp. 321-396. Oxford and New York:
Pergamon Press.
CAFFIER, P. (1929). Die proteolytische Fahigkeit von Ei und Eibett (Experimentelle Studien
mit Chorion und Dezidua). Zentbl. Gyna'k. 53, 2410-2425.
DENKER, H. W. (1969). Zur Enzym-Topochemie von Friihentwicklung und Implantation
des Kaninchens. Thesis, Med. Fak. Marburg (Germany) (see also Denker, 1971).
ANDARY,
748
H.-W. DENKER
H. W. (1910a). Topochemie hochmolekularer Kohlenhydratsubstanzen in Friihentwicklung und Implantation des Kaninchens. I. Allgemeine Lokalisierung und Charakterisierung hochmolekularer Kohlenhydratsubstanzen in fruhen Embryonalstadien. Zool.
Jb. (Physiol.) 75, 141-245.
DENKER, H. W. (19706). Topochemie hochmolekularer Kohlenhydratsubstanzen in Fruhentwicklung und Implantation des Kaninchens. II. Beitrage zu entwicklungsphysiologischen
Fragestellungen. Zool. Jb. (Physiol.) 75, 246-308.
DENKER, H. W. (1971). Enzym-Topochemie von Fruhentwicklung und Implantation des
Kaninchens, I, II, III. Histochemie 25, 256-267, 268-285, and 344-360.
DENKER, H. W. (1972). Blastocyst protease and implantation: Effect of ovariectomy and
. progesterone substitution in the rabbit. Acta endocr., Copenh. 70, 591-602.
DENKER, H. W. (1974). Protease substrate film test. Histochemistry (Histochemie) 38, 331338.
DENKER, H. W. & HAFEZ, E. S. E. (1974). Proteases and implantation in the rabbit: role of
trophoblast vs. uterine secretion. (In preparation.)
ENDERS, A. C. & SCHLAFKE, S. (1969). Cytological aspects of trophoblast-uterine interaction
in early implantation. Am. J. Anat. 125, 1-30.
ENDERS, A. C. & SCHLAFKE, S. (1972). Implantation in the ferret: epithelial penetration.
Am. J. Anat. 133, 291-316.
GRAFENBERG, E. (1910). Beitrage zur Physiologie der Eieinbettung. Z. Geburtsh. Gyna'k.
65, 1-35.
HALBAN, J. & FRANKL, O. (1910). Zur Biochemie der Uterusmukosa. Gyna'k. Rdsch. 4,
471^84.
KIRCHNER, C. (1972). Uterine protease activities and lysis of the blastocyst covering in the
rabbit. /. Embryol. exp. Morph. 28, 177-183.
KIRCHNER, C. (1973). Interferenzkontrastmikroskopische Untersuchungen uber die Lysis der
Keimhullen beim Kaninchen. Cytobiologie 7, 437^441.
KIRCHNER, C , HIRSCHHAUSER, C. & KFONKE, M. (1971). Protease activity in rabbit uterine
secretion 24 hours before implantation. /. Reprod. Fert. 27, 259-260.
MINTZ, B. (1971). Control of embryo implantation and survival. In: Schering Symposium on
Intrinsic and Extrinsic Factors in Early Mammalian Development, Venice 1970 (ed. G.
Raspe). Advances in the Biosciences 6, 317-342. Oxford: Pergamon Press; Braunschweig:
Vieweg.
MINTZ, B. (1972). Implantation-initiating factor from mouse uterus. In Biology of Mammalian
Fertilization and Implantation (ed. K. S. Moghissi & E. S. E. Hafez), pp. 343-356. Springfield, Illinois: Charles C. Thomas.
OWERS, N. O. (1970). Comparison of the proteolytic activity of the implanting rat and
guinea-pig blastocyst. Anat. Rec. 166, 358.
PEARSE, A. G. E. (1968). Histochemistry. Theoretical and Applied. 3rd ed., vol. 1, p. 665.
London: J. & A. Churchill.
SCHWICK, H. G. (1965). Chemisch-entwicklungsphysiologische Beziehungen von Uterus
zu Blastozyste des Kaninchens Oryctolagus cuniculus. Wilhelm Roux Arch. EntwMech.
Org. 156, 283-343.
SPEE, F. GRAF V. (1901). Die Implantation des Meerschweincheneies in der Uteruswand.
Z. Morph. Anthrop. 3, 130-182.
STEER, H. W. (1970). The trophoblastic knobs of the preimplanted rabbit blastocyst: a
light and electron microscopic study. /. Anat. 107, 315-325.
DENKER,
{Received 16 April 1974)