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/ . Embryol. exp. Morph. Vol. 24, 2, pp. 305-312, 1970
305
Printed in Great Britain
Coincidence of embryonic growth and uterine
protein in the ferret
By JOSEPH C.DANIEL, Jr.1
From the Department of Molecular, Cellular and Developmental Biology,
University of Colorado
SUMMARY
The protein content offluidsisolated from the lumen of the uterus of the ferret was correlated
with growth of the pre-implantation embryo. Similar observations were made with mink for
comparative purposes. Tt is concluded that in the ferret:
Protein content rises coincidentally with blastocyst expansion, becoming especially high
when the embryo is implanting.
The proteins present early in the period are of types that diffuse easily in electrophoresis;
they become more like those of serum near the time of implantation.
There is no evidence of an embryonic diapause. Mitotic activity in cells of the blastocyst is
maintained at a high rate (about 5 %).
The prolonged pre-implantation period results from a relatively long mitotic duration in
blastocyst cells (80+ min).
The protein content found during the pre-blastocyst period is similar to that of mink uterine
fluids collected during the period of embryonic diapause. This observation provides additional
circumstantial evidence that the embryonic diapause accompanying delayed implantation is
related to limited protein availability.
INTRODUCTION
Preliminary studies (Daniel & Krishnan, 1969) have shown that the fluids
taken from the lumen of uteri of those animals having obligate delayed implantation (e.g. black bear, fur seal, mink and armadillo) are low in protein
content and specifically lack any component comparable to the rabbit uterine
protein, blastokinin (Krishnan & Daniel, 1967), during the period when the
blastocysts are in diapause. In mink uterine fluids, taken near the end of the
diapause period (immediately preceding embryo 'reactivation' and subsequent
implantation), the amount of protein rises markedly and traces of a component
similar to blastokinin can sometimes be detected (Daniel, 1968; Daniel &
Krishnan, 1969). Thus, the reinitiation of blastocyst growth in mink parallels
quantitative (and possibly qualitative) changes in the uterine lumen proteins. It
is of interest to know whether uterine protein increases with embryonic growth in
the ferret {Mustek furo), a mustelid that is closely related to the mink (Mustek
vision) but does not have delayed implantation.
1
Author's address: Department of Molecular, Cellular and Developmental Biology,
University of Colorado, Boulder, Colorado 80302, U.S.A.
306
J. C. DANIEL
This paper reports the results of studies comparing the protein content of
ferret uterine fluid with stages of embryonic growth throughout the preimplantation and early post-implantation periods.
MATERIALS AND METHODS
Ferrets were made available to the author during the spring of 1969 from the
colony of John Hammond, Jr. of Cambridge University. Animals were selected
for breeding by the condition of the vulva. Duration of the pregnancy and age of
the embryos were measured from copulation as time zero. Animals were killed
by cervical dislocation on days 0, 1,3, 4, 5, 7, 8, 9, 10, 13, and 16 post coitum
(± 1 h), and embryos and uterine fluids collected at each of these times. Uteri
were removed from the animals. One horn was cut free at the cervical end,
the cut end blotted dry of blood on filter paper, and the horn then flushed with
1 ml of Tyrode's solution and the flushings collected into watch glasses. After
the contained embryos were removed to fresh saline, the flushings were centrifuged to remove cellular debris and then frozen for future analysis of their protein content (presumably, representative of the soluble proteins present in the
uterine lumen). The second horn was torn open with forceps, any obvious
embryos removed and added to those from the first horn, and then filter-paper
discs applied to the exposed endometrial surface to absorb the undiluted uterine
fluids. These discs were frozen and later used as the sample source for acrylamide
disc-gel electrophoresis. Exceptions to these methods were necessary for postimplantation stages where it was impossible to flush the embryos from the horn
or to get complete total samples of the fluids. In these cases, those portions of the
uteri that were free of a conceptus were cut away and serially flushed with the
same volume of saline. These additional cut surfaces increased the probability
of contamination of the sample with blood proteins but two animals, 13 and 16
days pseudopregnant, provided supplementary uncontaminated samples.
The isolated embryos were measured with an ocular micrometer and cell
number approximations made of pre-morula stages (less than 5 days p.c).
Acetic-orcein squash preparations were made of morula and blastocyst stages
to facilitate cell counts and determination of mitotic indices.
Total protein was determined spectrophotometrically by the Lowry procedure (Lowry, Rosebrough, Farr & Randall, 1951) compared to known sample
concentrations of bovine serum albumin. Two 0-5 ml aliquots of each sample
were measured and estimated as micrograms of protein per uterine horn.
Single determinations of the proteins were made of four animals that had only
degenerate eggs in their uteri after being respectively 7, 8, 13, and 16 days
pseudopregnant.
Uterine fluid samples collected on filter-paper discs were subjected to acrylamide gel electrophoresis in Tris-glycine buffer at pH 8-7. Runs were started at
60 V and increased gradually to 240 V with 5 mA/tube until the indicator dye
Embryonic growth and uterine protein
307
was about 1 cm from the bottom of the tube. Gels were fixed in 7 % acetic acid
and were stained in amido black for 3 h. They were then cleared in a series of
washes of 7 % acetic acid.
For comparison, similar samples were taken from two mink during the delay
period, namely 15 and 16 days/?os^ coitum, respectively, and measurements were
made in the same way as described above.
RESULTS
Table 1 lists the essential data from these studies. These include the uterine
protein determinations, embryo size, cell number and mitotic indices calculated
from the pooled cell counts of each stage.
Table 1. Embryonic growth and uterine protein content in the
ferret and the mink
Day
(p.c.)
0
1
4
5
7
7
8
8
9
10
13
13
16
16
15
16
No. and type of
embryos recovered
—
— (not yet ovulated)
16 cleavage stages
(from oviducts)
4 morulae, 3 degenerate eggs
9 blastocysts, 2 degenerate eggs
8 degenerate eggs
(pseudopregnant)
3 blastocysts, 8 degenerate eggs
17 degenerate eggs
(pseudopregnant)
14 blastocysts
12 blastocysts
17 implanted embryos,
1 degenerate egg
13 degenerate eggs
(pseudopregnant)
15 fetuses of 17-20
somites
1 degenerate egg
(pseudopregnant)
5 blastocysts, 2 degenerate eggs
8 blastocysts
Diam. of embryonic
vesicle (mm)
No. of cells
per embryo
Mitotic
index
Uterine
luminal
fluid
proteins
(/<g/horn)
Ferret
—
—
0-11-013 (0-12)*
—
—
2-12 (7)*
—
—
—
42-104
70-108
40-80
011-014 (0125)
8-60 (28)
—
60-109
0-2-0-25 (0-22)
107-182(146)
5-56
154-210
—
—
—
0-46-0-5 (0-48)
333-380 (356)
4-63
—
—
—
0-65-0-85 (0-77)
1-0-1-3 (118)
3-5-5-5 (4-7)
450-1106(705)
2000-4000J
—
5-59
5-26
—
—
—
—
—
—
2020-2280
—
—
—
11.10
Mink
0-35-0-4 (0-37)
153-188(171)
0
97
0-3-0-45 (0-38)
209-290 (257)
0-27
89
7-0-8-5 (7-8)
* Average value in parentheses.
158
180-200
156
156-280
690-758
1160-1400
820
t Estimated value.
E M B 24
308
J. C. DANIEL
The relationship between embryo growth and uterine protein is shown
graphically in Fig. 1.
The results of the acrylamide gel electrophoresis studies provided little
useful information. Except for albumin, which was obvious in all of the samples,
Embryo
2000
Protein
Pseudopregnant
Mink
1500 £
1000
500
0 1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
Days post coitum
Fig. 1. Early growth of the embryo and protein content in uterine luminal fluids of the
ferret. Protein values for mink and for pseudopregnant ferret are included for
comparison.
there was poor resolution of distinct banding in the patterns from uterine fluids,
an observation which suggests that most of the proteins may be of low molecular
weight and thus readily diffusing species. Samples taken on the ninth and
tenth days show some serum-like components but at no time was it possible to
demonstrate the band that had the mobility of rabbit blastokinin.
Embryonic growth and uterine protein
309
Fig. 2 plots cell replication of pre-implantation ferret embryos as a function of
time; it seems to show that no period of growth arrest or embryonic dormancy
exists in the ferret and provides the basis for explaining the relatively slow rate of
embryonic growth. It includes measurements extracted from the data of other
investigators.
Hammond & W a l t o n (1934)
Ovulation ,
.
Pronuclear eggs i
Fertilization > <•
1000-
>
Hamilton (1934) W/S->.
Marston & Kelly (1969) ]
Daniel
•
1
0
1
2
3
4
5
6
7
8
9
10
Days post coitum
Fig. 2. Cell replication in the ferret embryo previous to implantation. Data from
various investigators.
DISCUSSION
According to Hamilton (1934) 'incipient' blastocysts are first found in the
ferret on the sixth day post coitum, and Hammond & Walton (1934) in their
analysis of Robinson's (1918) data report blastula stages at 144 h. Marston &
Kelly (1969) found both morulae and blastocysts at 168 h p.c, and in Chang's
(1968) observations blastocysts were not found until day 7, only morulae on
day 6. Apparently, initial cavitation occurs sometime during the seventh day
310
J. C. DANIEL
(i.e. 144-168 hp.c). Implantation is on or about day 12 of the 40-43 day gestation period.
Fig. 1 demonstrates the close parallelism between blastocyst expansion and
the amount of protein present in the fluid of the uterine lumen (the parallelism
continues after implantation in relation to total embryo growth). During the
cleavage period when the ovum is relatively self-sufficient and little tissue differentiation is occurring, the protein level is negligible, but with blastulation it
increases and then rises dramatically with implantation. The variability in the
measurements makes is impossible to say with certainty that there is not a new
constant higher level of protein maintained for days 7 through 9 (as opposed to
a continuously rising level), but there is no doubt that the level is two to three
times higher than during the preblastocyst period. That the protein comes from
the genital tract rather than the embryo is shown by the higher concentrations
found in the pseudopregnant animals, where uteri do not contain viable embryos.
In the post-implantation pseudopregnancies the protein level is significantly
lower than in pregnancies of comparable stage. But this reflects a different
reproductive state, uterine size and vascularity, placental development, etc.
Buchanan (1966) has described the general growth of the uterine luminal
epithelium with steady increase in secretory products throughout the preimplantation period of the ferret, and the 'copious secretions' that accompany
hypertrophy of the epithelium during the implantation and post-implantation
periods. He concludes from histochemical evidence that the principal uterine
secretions are mucoproteins and glycoproteins. That these events are directly
related to the changes in the protein content of the uterine lumen seems very
likely.
The relatively long pre-implantation period of the ferret and slow growth of
its embryo lead one to suspect that an embryonic diapause of short duration
could exist in this mustelid. However, the information presented here discounts
that suspicion. In addition to the increasing protein content in the uterine fluids,
it is obvious that ferret blastocysts continue to grow and maintain a high level
of mitotic activity. Thus, there is no evidence of any obligate delay in implantation. (Recently, Buchanan (1969) has shown that a facultative delay
might be induced in the ferret because blastocyst expansion can be prevented by
ovariectomy on day 4 and implantation prevented by ovariectomy before
day 10.)
Reference to Fig. 2 will show that the continual increase in the number of
cells composing the embryo is the product of a doubling time of about 18 h.
Knowing the doubling time and the mitotic index, and assuming that cell death
is minimal and that mitosis is continuous rather than cyclic, it is possible to
calculate the mitotic duration from the formula of Smith & Dendy (1962):
M = -log e 2,
when M = mitotic index, T = mitotic duration and T = doubling time.
Embryonic growth and uterine protein
311
This calculation shows that the ferret blastocyst cells have a mitotic
duration of 80+ min. (For comparison, in the rabbit blastocyst where the
mitotic index is about 4-4 %, the cells double in 8 h because the mitotic duration
is only about 30 min.) Thus, prolonged mitosis seems to account for the somewhat slower growth of the pre-implantation ferret embryo.
When mink embryos, taken during the middle (15 and 16 days p.c.) of an
average diapause period of about 3 weeks duration, are compared to those of the
ferret, they are seen to be of a size intermediate between 7- to 8-day p.c. ferret
blastocysts, but exhibit essentially no mitotic activity. The protein content of the
uterine fluids of these mink is very low and at a level comparable to that found
in the ferret uteri between 0 and 5 days/?.c.: previous to the time of blastocyst
development! Earlier studies from this laboratory (Daniel & Krishnan, 1969),
though not reporting absolute measurements of mink uterine protein, did
nevertheless note that it was very low (in all animals tested with delayed implantation) and that it increased fourfold or more just before embryo activation
and subsequent implantation. Such an increase would provide a level not unlike
that reported here during implantation of the ferret. These observations support
the hypothesis that the embryonic diapause accompanying delayed implantation results from a uterine condition where inadequate protein is available
to the blastocyst.
Chang (1968) has demonstrated that mink blastocysts transplanted to ferret
uteri will become 'activated' and grow, while ferret blastocysts become 'dormant1 and eventually die in mink uteri. He has further shown that hybrid embryos, resulting from ferret ova fertilized by mink sperm, do not experience
a diapause when they reach the blastocyst stage in the ferret uterus (Chang, 1965).
These studies support the hypothesis expressed above, in that, although they
do not relate directly to uterine protein, they clearly show that some condition of
the uterus, and not some inherent quality of the embryo alone, is responsible for
the arrested development of mustelid blastocysts in cases of delayed implantation.
RESUME
Coincidence de la croissance embryonnaire et de la proteine uterine chez le furet
Le contenu protemique de liquides extraits de la cavite uterine du furet a ete mis en correlation avec la croissance de l'embryon en pre-implantation. Des observations similaires ont ete
faites, a titre de comparaison, chez le vison. II est conclu que chez Je furet:
Le contenu protemique s'accroit parallelement avec 1'expansion du blastocyste, s'elevant
particulierement lorsque l'embryon s'implante.
Les proteines presentes au debut de la periode sont de types qui diffusent facilement en
electrophorese; elles deviennent plus semblables a celles du serum au moment de Pimplantation.
II n'y a aucune evidence d'une diapause embryonnaire. L'activite mitotique dans les cellules
du blastocyste reste elevee (a peu pres 5 %).
La periode prolongee de pre-implantation resulte d'une longue duree de la mitose (80+ min)
des cellules du blastocyste.
La teneur en proteines trouvee au cours de la periode pre-blastocystaire est semblable a
celle des liquides preleves dans 1'uterus de vison au cours de la periode de diapause embryon-
312
J. C. DANIEL
naire. Cette observation apporte de nouvelles preuves circonstanciees en faveur d'une relation
entre la diapause accompagnent Povulation differee et la disponibilite limitee en proteines.
The author wishes to express his gratitude to John Hammond Jr. for supplying the animals
used in this study, and for his constructive criticism of the manuscript, to Dr C. R. Austin and
Dr D. A. T. New in whose laboratories the work was done, and to Dr John Marston,
Mrs Pat Coppola and Mrs Venitha Dharmawardena for technical assistance. The work was
supported in part by NSF Grant no. GB-6363, AEC Contract no. AT (11-1)-I597, and a
faculty fellowship to the author from the University of Colorado.
REFERENCES
G. D. (1966). Reproduction in the ferret (Mustelafuro). I. Uterine histology and
histochemistry during pregnancy and pseudopregnancy. Am. J. Anat. 118, 195-216.
BUCHANAN, G. D. (1969). Reproduction in the ferret (Mustela furo). II. Changes following
ovariectomy during early pregnancy. /. Reprod. Fert. 18, 305-316.
CHANG, M. C. (1965). Implantation of ferret ova fertilized by mink sperm. /. exp. Zool. 160,
67-70.
CHANG, M. C. (1968). Reciprocal insemination and egg transfer between ferrets and mink.
/. exp. Zool. 168, 49-60.
DANIEL, J. C. (1968). Comparison of electrophoretic patterns of uterine fluids from rabbits
and mammals having delayed implantation. Comp. Biochem. Physiol. 24, 297-300.
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components and the diapausing state of blastocysts from mammals having delayed implantation. /. exp. Zool. 172, 267-282.
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HAMMOND, J. & WALTON, A. (1934). Notes on ovulation and fertilization in the ferret. /. exp.
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(Manuscript received 29 September 1969)