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/. Embryo!, exp. Morph. Vol. 31, 2, pp. 479-487, 1974
Printed in Great Britain
479
Micro-disc electrophoresis of soluble proteins
in rabbit blastocysts
ByULRICH PETZOLDT1
From the Arbeitsgruppe Prof. Gottschewski am Max-Planck-lnstitut
fiir Immunbiologie
SUMMARY
Soluble proteins were studied in the developing rabbit blastocyst by using a micro-disc
electrophoresis technique. The tissues of the blastocyst (in later stages separated into the
embryoblastic and trophoblastic parts) were analysed, and their protein patterns were compared with those of uterine secretion and blastocyst fluid.
A protein pattern with more than 20 protein fractions was found in the tissues of the blastocysts. This protein pattern was quite different from those previously reported in uterine
secretion or blastocyst fluid. After cleavage and during early phases of development of blastocysts the protein pattern was changing; from the 5th day p.c. it became fairly homogeneous.
The protein patterns of the embryonic and trophoblastic parts of the blastocyst tissues were
rather similar.
The origin of the proteins in the blastocyst fluid is discussed.
INTRODUCTION
Intensive studies have been performed to identify the protein components in
different fluids of the genital tract of the rabbit (Schwick, 1965; Beier, 1968;
Urzua, Stambaugh, Flickinger & Mastroianni, 1970; Shapiro, Jentsch & Yard,
1971). Remarkable changes in the protein pattern of the uterine fluid were
reported early in pregnancy (Kirchner, 1969) and following hormonal administration (Beier, 1968; Kirchner, 1971). Much is also known about the protein
content of the blastocyst fluid (Kirchner, 1969; Hamana & Hafez, 1970), and
protein synthesis in cleaving embryos and blastocysts (Monesi & Salfi, 1967;
Manes & Daniel, 1969; Tasca, 1969). On the other hand, few studies are available
on the protein pattern of the embryonic cells (Manes & Daniel, 1969; Petzoldt,
Dames, Gottschewski & Neuhoff, 1972; Petzoldt, 1972). While the protein
content of the mouse egg is reduced throughout the preimplantation period
(Brinster, 1967 0), in the rabbit the protein content of embryos increases considerably during blastocyst formation (Hafez & Sugawara, 1968). So there is a
better chance of studying protein pattern in the rabbit embryo during this time.
This paper is concerned with analysis of soluble proteins in rabbit embryos
from morula to late blastocyst stages using micro-disc electrophoresis. It also
1
Author's address: Arbeitsgruppe Prof. Gottschewski am Max-Planck-Institut fur Immunbiologie, 78 Freiburg i. Br., Stefan-Meier-Strasse 8, Germany.
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U. PETZOLDT
shows preliminary trials of separating embryoblastic and trophoblastic parts of
the blastocyst, comparing their protein patterns to each other and to the surrounding fluids.
MATERIALS AND METHODS
Three- and four-day-old embryos were collected from the oviducts and uteri
of superovulated rabbits. Superovulation was induced according to the following
methods. (1) 150 i.u. pregnant mare serum gonadotrophin (Schering AG) were
injected intramuscularly and were followed 96 h later by an intravenous (i.v.)
injection of 100 i.u. Prolan ® (Bayer, Leverkusen) and normal mating (modified
from Brinster, 19676). (2) 0-33 mg follicle stimulating hormone (Armour
Pharmaceutical Co.) was injected intramuscularly daily for 4 days. The
FSH was dissolved in 1 ml of 20 % solution of Kollidon 25. Normal mating on
the fifth day was followed by an i.v. injection of 100 i.u. Prolan ® (modified from
Petzoldt, Briel, Gottschewski & Neuhoff, 1973). Natural matings without
hormonal treatment were used to study embryos of later developmental age.
Autopsy was done 3, 4, 5, 6 days p.c. ± 1 h and 6 days 17 h p.c. ± 1-J- h. Each
uterine horn was immediately flushed with 2 ml of cold Hanks' medium (pH 7-27-5, without phenol red but including 0-01 M-NaF); 3-day p.c. oviducts were
flushed with 1 ml of the medium. The fluid was centrifuged and stored in a deepfreezer.
The embryos were washed several times in cold Hanks' medium. The 3-day
eggs covered with their oolemma and mucolemma were enclosed in microcaps
(Petzoldt et al. 1972). Blastocysts were punctured after washing and the blastocyst
fluid was withdrawn and enclosed in a microcap. Blastocyst tissues were separated
from the coverings, and both were enclosed after washing in Hanks' solution in
microcaps and stored in the deep-freezer. In 6-day and 6-day 17 h embryos the
blastocyst tissues also were separated into the embryonic and trophoblastic
constituents using sharp steel needles. Unfortunately it was not possible to get
the embryonic disc free of lining trophoblastic cells and 'Rauber's layer'.
The protein pattern was analysed by using micro-disc electrophoresis (Hyden,
Bjurstam & McEwen, 1966; Neuhoff, 1968; Neuhoff & Schill, 1968) 'ml (A or
5 /A microcaps as was done in earlier studies (Petzoldt et al. 1972). Flushed
uterine secretion and blastocyst fluid could be analysed directly; 3-days p.c.
morulae and embryonic tissues were homogenized in the microcaps by repeated
freezing and thawing in Hanks' solution. Coverings were homogenized in a
microcap with a nerve-channel drill at 12000 rev/min for 15 sec; they were also
frozen and thawed several times. After centrifuging (1 h, 15000 rev/min, in a
refrigerating chamber) the supernatant fluid was used for analysis.
Nearly 20 embryos 3 days old and the tissues of nearly ten blastocysts 4 days
old were necessary to have a good micro-disc electrophoresis in a 2 /A microcap.
Tissues of one 5-day embryo, 1 embryoblastic disc of a 6-day blastocyst and half
an embryoblastic disc of a 6-day 17 h blastocyst were enough for an electro-
Soluble proteins in blastocysts
481
phoresis in a 5 p\ microcap. From trophoblastic tissues of one 6-day or 6-day
17 h blastocyst one could make several analyses in 5 fi\ microcaps.
The gels were stained in an amido black solution (0-5 %) and fixed in acetic
acid (7-5 %). Densitometric evaluation was done with a Joyce-Loebl Doublebeam Microdensitometer. For planimetriation the curves were divided into
groups of bands and single bands, and their percentage portion of the total
protein was counted from 8-12 curves. These values were compared statistically
in the different developmental stages.
RESULTS
The protein patterns of oviducal, uterine (Fig. 2 c) and blastocyst (Fig. 2/)
fluids and their changes in several developmental stages were known from previous studies and were only used here to compare protein patterns of the blastocyst tissues. It should be pointed out that in the micro-disc electrophoresis,
uteroglobin will run in front of albumin due to its lower molecular weight
(Petzoldt et al. 1972; Murray, McGaughey & Yams, 1972). Blastocyst fluid had
a protein pattern similar to uterine secretion in the corresponding age of
pregnancy. This pattern was also seen in 4-day-old embryos, which has not been
reported before.
In contrast to all these fluids the tissues of the embryo had their own specific
protein pattern with 20 and more protein bands. Since evidence is still unavailable
concerning these proteins and their immunological identity to other proteins, it
is difficult to compare the protein patterns of several developmental stages and to
recognize quantitative and qualitative changes.
At 3 days p. c. protein patterns were variable from one microgel to the other and
there was no uniformity in several analyses of the same embryonic age. There
were two main groups of protein patterns in this stage—one with the dominant
bands at the slowly migrating protein fraction (Fig. 1 a), the other with a very
sharp band after a third of the running distance (Fig. \b). However, at 3 days
p.c, these differences could be attributed to the recovery of eggs at variable
developmental phases. At this stage the majority of recovered eggs were morulae
with 16-128 cells, while others were blastocysts in their early phases of development, showing a small blastocyst cavity. Eggs were located partly in oviducts and
partly in uteri.
There was a minor relationship between the protein patterns of 3-day-old
embryos and 4 days p.c. blastocysts (Fig. 1 c). The variation of gels was smaller at
the 4-day stage than in the morula stage. The protein pattern was more related to
that of the later blastocyst stages than to that of the 3-day eggs, but it was still
different. These quantitative changes of the protein fractions analysed from the
3- to 5-day-old embryos were probably accompanied by qualitative changes.
In the tissues of 5-day blastocysts (Fig. 1 d) we found a rather homogeneous
protein pattern which is consistent till the 6-day 17 h stage (Fig. 1 e,f). Certainly
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U. P E T Z O L D T
(b)
Fig. 1. Protein patterns (pherograms and gels) of soluble proteins in rabbit morulae
and blastocyst tissues, (a) 3 days^.c, (b) 3 daysp.c, (c) 4 daysp.c, (d) 5 daysp.c, {e) 6
daysp.c, (/) 6 days 17 hp.c. Arrows in (c)-(/) show the electrophoretic mobility of
rabbit serums albumin (A) and uteroglobin (U) when they were added to blastocyst
tissue homogenate. (The different amounts of proteins and staining intensities in
the different gels were compensated by using different neutral wedges for the
densitometric evaluation.)
Soluble proteins in blastocysts
483
i\ fa
Uteroglobin
Albumin
Uteroglobin
-Glycoprotein
t
I ^
Fig. 2. Protein patterns (pherograms and gels) of soluble proteins in separated
blastocyst tissues, uterine secretion and blastocyst fluid, (a) Embryoblastic tissues
6 days p.c. (b) Embryoblastic tissues 6 days 17 hp.c. (c) Uterine secretion 6 days/>.c.
(d) Trophoblastic tissues 6 days jP.c. (e) Trophoblastic tissues 6 days 17 h p.c. (/)
Blastocyst fluid 6 days p.c. (see Fig. 1).
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U. PETZOLDT
there were some quantitative changes inside a single developmental stage from
one gel to the other, and also one can sometimes see differences among the protein patterns of the several developmental stages, but counting the curves from
5 days p.c. to 6 days 17 hp.c. we could not see changes in single protein bands
or groups of bands which would be dependent on the blastocyst development.
Fig. 2 shows gels and curves of 6 days p.c. separated embryoblastic (a) and
trophoblastic (d) tissues and 6 days 17 h p.c. separated embryoblastic (b) and
trophoblastic (e) tissues. All these curves were also related to each other. In both
stages one could see quantitative differences between some protein groups in
embryoblastic and trophoblastic tissues, but there seemed to be some other
differences between 6 days/?.c. and 6 days 17 hp.c. We cannot say at the moment
if there are real differences in protein bands discernible with the technique of
micro-disc electrophoresis.
As in cleaving eggs (Petzoldt et al. 1972), the main quantity of proteins belonged
to the slower migrating fractions, in contrast to the surrounding fluids, where
albumin and uteroglobin with lower molecular weight were the most frequent
proteins.
The identification of these proteins is still rather difficult. Addition of known
proteins of the uterine secretions, uteroglobin or rabbit serum albumin to the
blastocyst tissue homogenates showed that they were migrating with the same
velocity as special bands of the blastocyst protein (Fig. 1 c-f, ' A ' and 'U'). Both
proteins were very important in the surrounding secretion (Fig. 2 c) and the
blastocyst fluid (Fig. 2/). The similarity of the electrophoretic mobility has not
yet answered the question of immunological identity. Preliminary immunological
studies show immunological identities between blastocyst and uterine proteins.
Further experiments are still in progress in the author's laboratory to solve this
problem.
In the isolated coverings of the blastocyst one could sometimes find slight
protein bands, but it is hard to decide whether these bands have resulted from
proteins in the uterine secretion or blastocyst tissues adhering to the sticky
coverings. Moreover, homogenization in only Hanks' medium is probably not
effective enough for dissolving covering proteins.
DISCUSSION
The formation of the blastocyst in rabbits shows two different events: on one
hand the formation of the blastocyst fluid within the blastocyst cavity, and on the
other hand the augmentation of the embryoblastic as well as the trophoblastic
parts of the embryo. Concerning the protein analysis of blastocyst tissues and
fluid, different protein patterns for those materials could also be seen.
(1) In the embryonic tissues after cleavage and at the beginning of blastocyst
formation we see a remarkable increase of protein synthesis in harmony with an
increase of RNA synthesis in the embryo (Monesi & Salfi, 1967; Manes &
Soluble proteins in blastocysts
485
Daniel, 1969; Manes, 1969). Likewise, in the protein analysis there is a change in
the protein pattern in quantity and probably in quality. These events are nearly
simultaneous with the passage of the embryo from the oviduct to the uterus and
thus in the change of the protein surroundings. In spite of the rapid growth and
huge accumulation of proteins in developing blastocysts, no visible changes were
observed in their protein pattern.
(2) Protein patterns in 4-, 5-, 6- and 6|-day blastocyst fluids were similar to the
corresponding patterns of uterine secretion samples (Kirchner, 1969; Hamana
& Hafez, 1970).
This demonstrated that the proteins inside the blastocyst were similar to those
of the surrounding fluids, whereas the tissues dividing both media showed a
totally different protein pattern. Relation of some tissue proteins to uterine and
serum proteins was possible, but not with certainty.
There is still an undecided point whether the proteins of the blastocyst fluid
are synthesized by the blastocyst tissues or whether active transport of proteins
takes place from the uterine fluid to the blastocyst fluid before implantation. The
transport of proteins could be supported by the following evidence:
(a) Non-specific proteins were recovered from the blastocyst fluid, when they
were added to culture medium in vitro (Beier & Maurer, 1973).
(b) Using immunohistochemical techniques, Kirchner (1972) was able to
prove that uteroglobin is transported across 'channels', which penetrate through
the coverings of the blastocyst.
At the moment we prefer the opinion that most blastocyst fluid proteins are
derived from uterine secretion proteins and not synthesized by the embryo itself.
The functions of the uterine protein for the blastocyst are probably various:
nutrition of embryos (Beier, 1968; Kirchner, 1969); inducer functions for the
formation of blastocyst (Krishnan & Daniel, 1967), and also hormone binding
functions (Urzua et ah 1970; Wiest & Rao, 1971; Arthur, Cowan & Daniel,
1972). However, these would not exclude the principle that maternal proteins of
the uterine secretion and blastocyst fluid will mask the immunologically strange
embryo and its protein, as has been suggested for implantation (Zimmermann,
1965,1966).
The study of protein patterns of the blastocyst tissues during blastocyst
formation has also indicated very important changes inside the embryo. Such
changes could be, for example, the conversion of metabolic pathways (Fridhandler, 1961; Brinster, 1968). Concerning the protein patterns it was difficult to
find differences during the later blastocyst growth and to differentiate between the
embryoblastic and trophoblastic parts of blastocyst tissues. This was certainly
due, in part, to the difficulties of tissue preparation, but it is also possible that
minor changes in some protein bands, e.g. enzymes, were invisible with our
technique of micro-disc electrophoresis. Combined methods of immunoelectrophoresis and autoradiography could settle these difficulties and give better
results.
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U. PETZOLDT
The author is grateful for the excellent technical assistance of Miss Doris Gensmantel and
for the revision of the English text by Dr I. Aref. The work was supported by the Deutsche
Forschungsgemeinschaft, Pe 166/3, Go 65/15. The uteroglobin used was isolated by Dr
Chr. Kirchner, Zoologisches Institut, Marburg.
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