/. Embryol. exp. Morph., Vol. 17, 2, pp. 293-302, April 1967
With 2 plates
Printed in Great Britain
293
Studies on growth of the mink blastocyst
By JOSEPH C.DANIEL, J R 1
From the Institute for Developmental Biology, University of Colorado
INTRODUCTION
In those mammals in which implantation is delayed, the embryo enters a
diapause at the blastocyst stage. The present report describes experiments with
mink over the last three breeding seasons, attempting to define the factors that
limit development at this stage.
METHODS AND MATERIALS
Four approaches to the problem were used: (1) determination of growth of
mink blastocysts in vitro with specific modifications of media; (2) transplantation
of mink embryos to rabbit uteri; (3) direct treatment of pregnant female mink
with ergosterol; (4) growth of rabbit blastocysts in vitro in medium containing
mink serum.
(1) The culture methods used for mink embryos were those developed for the
rabbit and described in earlier publications (Daniel, 1963, 1965). Mink blastocysts (Plate 1) were isolated in culture medium after being flushed from the
uteri of mothers bred 9-20 days earlier. Various components were added to the
medium, F10 (Ham, 1963), in concentrations that were previously tested
against rabbit blastocysts and found to be non-toxic, and, in some cases, beneficial to growth. Rabbit uterine fluids were obtained for addition to the culture
medium by soaking, in 1 ml of F10, five filter paper discs that had absorbed the
fluid when placed against the moist endometrium of a freshly opened rabbit
uterus on the fourth day post coitum. In one experiment the mink blastocysts
were exposed to medium containing pronase until the zona was digested (or
reduced to a thin membrane), and then removed to pronase-free medium for
culture. In another case F10 was not used but rather the fluid from inside the
blastocysts of 7-day pregnant rabbits. The actual components tested are listed
in Table 1. In all cases growth was determined by measurements made with an
ocular micrometer.
(2) In the hope of providing a growth-supporting uterine environment, mink
blastocysts removed from the mother on days 10, 13 or 15 post coitum were
1
Author's address: Institute for Developmental Biology, University of Colorado, Boulder,
Colorado, U.S.A.
294
J. C. DANIEL, JR
transplanted to the uterus of a rabbit that had been mated 3 days before the
surgery. The uterotubal junction on the left side was ligated to prevent rabbit
embryos from reaching the uterus and the cervical end was also ligated (on the
last two rabbits in the series) to prevent expulsion of the mink blastocysts from
the vagina. The right side was left unaltered to act as a control. All the blastocysts (6-9) from one mink were removed, after being measured, from the
flushing dish in glass capillary tubes and then blown from the tube through
a small hole near the oviductal end of the uterus into the uterine lumen.
Standard closure and postoperative care were exercised. After 2 days the rabbits
were sacrificed and both mink and rabbit embryos flushed from their respective
horns. The mink blastocysts were measured again to establish whether growth
had occurred and the rabbit blastocyst was compared with the normal growth
stage of 5-day post coitum embryos.
(3) The possible effect of ergosterol on shortening the gestation period was
tested. Ten mink, bred on the same day, were divided into two groups of five
animals each. The animals of one group were each given 7500 i.u. of ergosterol
in corn oil intramuscularly on days 12, 17 and 22 of pregnancy. In the other
(control) group the animals were sham-injected with corn oil alone on the same
days. All were supplemented with vitamins A and E. The gestation period and
number of young for each were recorded.
(4) To test whether some factor exists in mink which might inhibit blastocyst
growth, rabbit blastocysts were cultured in media supplemented with mink
serum. Supplementation was at the level of 1, 5 or 10%. The experiments were
repeated with serum previously dialyzed against F10 for 24 h at 4 °C. The F10
dialysate was also used with 10% rabbit serum supplementation.
RESULTS
The results of the studies attempting to grow mink blastocysts in vitro are
listed in Table 1. It will be noted that the only specific factors, of those tested,
that produced any measurable growth in the majority of the specimens under
culture conditions were ergosterol and estrone, but this growth was not sustained
for any longer than 18 h.
DISCUSSION
The length of gestation in the mink varies from 42 to 79 days with an average
of 51 days, implantation occurring about 28-30 days before parturition (Enders
& Enders, 1963). Allowing most of 2 days between coitus and fertilization
(Enders, 1952) and 4 or 5 more days for cleavage of the ovum up to the blastocyst stage (Hansson, 1947), the delay in implantation of the mink blastocyst
averages about 14-17 days. The blastocyst is not completely dormant during
this period but the growth rate is very slow (Text-fig. 1), and is possibly the
product of cell expansion and fluid uptake rather than cell replication (see
1%
2%
5%
io;%
15%
50%
5%
10%
25%
0-5% in F10 for 3 min
Each at 1 mg/cc
Each at 01 mg/cc
Concentration
3
6
5
4
8
2
8
8
4
5
5
8
No. of
embryos
used
10,20
9, 16
13, 15,16
9,10, 16
10,16
10
10
9, 14,17
11
Age of embryos
(days p.c.)
.1
++
Growth
6, 18
4,12
Duration of growth
(hours)
1
9, 16
10
Zona removed with pronase
Mixture of carbohydrates*
13, 17
6,6
15
Mixture of 13 metabolic
intermediates!
|
Estrone(+10% R.S.)
12, 18
3
2/ig/cc
9, 16
S'
Ascorbic Acid ( + 1 0 % R.S.)
10
10"5 M
4
12
Ergosterol ( + 1 0 % R.S.)
10~7 M
12, 12, 12
5
10, 16
Tocopherol ( + 1 0 % R.S.)
10~5 M
12
10
4
17
4
7-day rabbit blastocyst fluid
4
10, 16
Rabbit uterine fluid in
Contents of 5 discs
F10R10
4
11
Anginase
0-2 mg/cc
Glyceraldehyde phosphate
6
0-2 mg/cc
11, 15,17
dehydrogenase
0-2 mg/cc
3
14, 17
Alkaline phosphatase
0-2 mg/cc
Diaphorase
2
14
0-2 mg/cc
DPNase
3
14
0-2 mg/cc
/?-glucuronidase
6
13, 15
0-2 mg/cc
Malate dehydrogenase
6
13, 15
0-2 mg/cc
Lactate dehydrogenase
6
13, 15
Mixture of enzymes listed above Each at 0-2 mg/cc
8
13, 14,15
"T" — — """"" *"~
~~~
* Glycogen, fructose, galactose, lactose.
t Glucose, D-glucose-6-phosphate, D-fructose-1, 6-diphosphate, phospho-enol-pyruvate, acetyl-coenzyme A, citrate, D-isocitrate, succinate, malate, lactate,
acetate, 6-phosphogluconate.
Rabbit serum
F10 alone
Mink serum (maternal)
Component or condition
tested (FlOx)
Table 1
296
J. C. DANIEL, JR
Table 2. Results of culturing 5-day-old rabbit blastocysts in media containing
mink serum or serum dialysate
Medium composition
F10
F10
F10
F10
F10
F10
F10
Growth for 24 h
alone
with 10% rabbit serum
with 1 % mink serum
with 5 % mink serum
with 10% mink serum
with 10% dialyzed mink serum
dialysate with 10% rabbit serum
+++++
+++++
++++ "1
JJ
+++++
Comments
Collapse within 12 h
Table 3. Results of transplanting mink blastocysts to rabbit uteri
on third day post coitum
Age of mink
blastocysts
No.
transNo.
planted recovered
10 days
7
1
13 days
9
7
15 days
6
5
Condition of
control rabbit
blastocysts
Condition of recovered embryos
Collapsed but partially reconstituted after 6 h in vitro; some
growth of zona
No growth; cells accumulating
into masses; dark
Collapsed; dark
Normal
Normal
Normal
Table 4. Results of administering ergosterol to mink during the
embryonic diapause
All were bred on 17 March. Injections were made on 29 March, 3 April and 8 April.
Experimental details in text.
Animal no.
1
2
3
4
5
6
7
8
9
10
Day of whelping
Gestation time
(days)
Experimental
5 May
7 May
14 May
25 May
Not
Average
49
51
58
69
—
56-75
Controls (sham-injected)
5 May
49
7 May
51
11 May
55
12 May
56
15 May
59
Average
54
No. of kits
4
7
6
1
—
4-5
5
6
9
3
4
5-4
Growth of mink blastocyst
297
Baevsky, 1963). The typical mink blastocyst requires some 2 weeks to achieve
the same size to which a rabbit blastocyst will grow in one day. Essentially, the
problem at hand concerns the cause of the reduction of rate of growth of the
mammalian blastocyst in species where implantation is delayed, as compared
to growth in those where there is no delay.
There have been many studies of the role of the maternal organism during
delayed implantation but they have not succeeded in clarifying the problem in
relation to the embryo. Ultimately, these two aspects of the same problem
cannot, of course be separated, but for investigational purposes it seems desirable
to divorce the living blastocyst from the maternal influence and study it in vitro.
Enders & Pearson (1946) have already used this in vitro approach on mink
embryos with negative results. However, because of the improvements in
embryo culture techniques since that time, and because of some encouraging
results with fur-seal embryos (Daniel, 1967), it was decided to utilize this approach
for some aspects of our attempt to define the factor(s) that limit development
of diapausing blastocysts.
The culture medium F10 is a defined medium which has been used alone, or
with small serum supplementation, to support good growth of a variety of
mammalian cells and some embryos in vitro. If mink blastocysts entered diapause
because of some common nutritional deficiency of the uterine fluids, one could
expect these embryos to grow when that deficiency was corrected by a good
culture medium. Our first efforts, therefore, involved the use of F10 supplemented with different proportions of mink (maternal) or rabbit serum. As
noted in Table 1, no consistent growth was obtained. What little success was
achieved occurred when mink serum was at the 1-2 % level. Since F10 would
not support growth, it was suspected that a factor in mink serum, when kept at
very low concentrations, might permit some growth, but at higher concentrations
would be detrimental.
To test for the existence of some factor which could inhibit blastocyst
growth, rabbit blastocysts, 5 days post coitum, were cultured in F10 with
varying amounts of mink serum, rabbit serum, dialyzed mink serum, or mink
serum dialysate with rabbit serum. As shown in Table 2, rabbit blastocysts
could not grow in medium containing mink serum in concentrations above 1 %,
which might indicate the existence of a factor inhibiting blastocyst growth in
the mink. This factor would appear to be non-dialyzable. The identical experiment
was performed using fur-seal serum with the same results (Daniel, 1967), except
that even the serum dialysate inhibited rabbit blastocysts. One may conclude
that the factor in question might be a macromolecule in the mink but a small,
dialyzable molecule in the fur-seal. There are probably two or more different
factors involved.
The transfer of mink blastocysts to the rabbit was an attempt to provide the
type of uterine environment that normally supports rapid blastocyst growth for
blastocysts that apparently do not normally enjoy this environment. Positive
19-2
298
J. C. DANIEL, JR
20
1-9
1-8
17
1-6
1-5
1-4
1-3
1 2
"
M
0 9
'
0-8
07
06
0-5
0-4
0-3
0-2
0-1
1 2
3 4 5 6 7 8 9 10 1 1 1 2 13 1415 16 17 18 19 20 2122 23 24 25 26
Days post coitum
Text-fig. 1. Growth of the mink blastocyst (area enclosed by light lines) compared
with that of the rabbit (heavy line). O, Specimens used in the present study; #,
from the data of Baevsky (1963); D, Hansson (1947); • , Enders (1938).
Growth of mink blastocyst
299
results would have established the need of some growth factor(s) available in
the uterine fluid. Unfortunately, negative results were obtained, indicating only
that the blastocysts of the mink do not find the uterus of the rabbit favourable
to their survival (see Table 3). Chang (1966) has reported similar results from
his transplants of ferret blastocysts to rabbit uteri.
To look for growth-promoting factors in vitro, the medium was first supplemented with various agents with which some fur-seal blastocyst growth had been
achieved. These included rabbit uterine fluids, estrone, ascorbic acid, and lactose
(used here in combination with some other carbohydrates). Of these only
estrone gave some positive results with mink blastocysts (see Plate 1, D, E). The
growth of these blastocysts in estrone medium was slow and of short duration,
however, so that it is most likely the result of fluid uptake rather than accelerated
cell division, as demonstrated with fur-seal blastocysts under the same conditions
(Daniel, 1967).
In the earlier paper, the rationale behind the use of an estrogen in the experiments with blastocysts in diapause was described, as well as the hypothesis that
some product of the action of estrogens on the uterine endometrium might be a
necessary factor for blastocyst growth. A number of enzymic activities have
been reported to increase in the endometrium under estrogen activation.
Among these /?-glucuronidase (Leatham, 1959), DPN-diaphorase (Rosa &
Velardo, 1959), malic dehydrogenase (Barker, 1964), lactic dehydrogenase
(Bever, 1959), alkaline phosphatase (Leatham, 1959), and carbonic anhydrase
(Lutwak-Mann, 1955). Except for carbonic anhydrase, which was tested earlier on
fur-seal blastocysts, each of these and a few others whose presence was suspected
were used singly in the culture medium, and then all of them combined, but in
no case was any growth observed in vitro. If the diapause is related to some
product of the estrogen-activated endometrium, it is other than those tested
here. Cochrane & Shackleford (1962) have reported that in vitro estrogen
injections do not shorten the gestation period in the mink.
Because the gestation period in some mustelids (mink included) can be
shortened by increased exposure to light, it was thought that possibly some
light-activated factor might be needed and ergosterol was suspected. Mink
blastocysts grew for short periods in ergosterol-supplemented medium but this
growth could not be prolonged by repeated changes of medium. However,
because of the initial success with this compound in vitro, animals were injected
on the schedule described above to see if supplementation of this vitamin
would overcome, or reduce the length of, the delay. There was no significant
difference in the gestation times or the litter size between these experimental
animals and the controls (Table 4).
Earlier work has shown that the mammalian embryo probably does not have
complete aerobic respiration before the late morula to early blastocyst stage
(Fridhandler, 1961), but can utilize progressively more respiratory metabolic
intermediates as growth proceeds throughout cleavage (Brinster, 1965).
300
J. C. DANIEL, JR
It was thought therefore that mink embryos might grow when provided
with an energy source besides the glucose and pyruvate found in the medium.
In view of the large number of possibilities, and the limitations on numbers of
available blastocysts, two media were utilized. One of these represented combinations of sugars (plus glycogen), and the other utilized a group of intermediates,
all of which will support growth of rabbit blastocysts. Neither of these combinations proved useful in promoting growth of diapausing mink blastocysts.
The growth retardation is therefore not believed to be the product of any
deficiency of respiratory enzymes or special energy source requirements.
Although not a part of the experimental plan, one other observation merits
note. In at least twelve cases the blastocysts collapsed while being flushed from
the uterus, but when retained in vitro all but two of these reconstituted by
filling with fluid (see Daniel, 1963). Since blastocyst growth is the product of
cell replication and fluid accumulation, and since observation of reconstitution
would seem to demonstrate that the trophoblast cells of diapausing blastocysts
are capable of their fluid-provision function, then the growth failure must be
related to the fact that the cells are not actively replicating. This is supported by
the work of Baevsky (1963), who has shown that there is little or no mitotic
activity in diapausing mink blastocysts. As the rate of cell replication is also
extremely low or non-existent in most other diapausing blastocysts, further
experiments will be designed to seek the cause of delayed implantation in the
control of mitosis.
SUMMARY
Mink blastocysts grew in vitro when the culture medium contained estrone or
ergosterol, but the growth was slow and of short duration, and believed to be
the product of fluid uptake rather than cell proliferation.
Ergosterol failed to shorten the gestation period when administered to mink
during the delay phase of pregnancy, the same failure having been reported
earlier with estrogen.
Mink blastocysts did not grow in vitro in medium containing other vitamins,
enzymes known or suspected to be present in uterine fluids, a variety of energy
sources, rabbit uterine or blastocyst fluids or when transplanted to the uterus
of 3- to 4-day pregnant rabbits.
A factor inhibiting blastocyst growth is believed to exist in the mink because
rabbit blastocysts cease growing and collapse when retained in culture medium
whose mink serum component is 5 % or higher. Dialysis does not remove the
factor.
Collapsed mink blastocysts can reconstitute to their original fluid-filled state
in vitro.
Growth of mink blastocyst
301
RESUME
Recherches sur la croissance du blastocyste de vison
Des blastocystes de vison croissent in vitro quand le milieu de culture contient
de l'oestrone ou de l'ergosterol, mais la croissance est lente et de courte duree, et
on pense qu'elle est le resultat d'une absorption de liquide plutot que d'une
proliferation cellulaire.
L'ergosterol n'a pas raccourci la gestation quand on l'a administre a des
visons pendant la phase de retard de la gestation; la meme absence de resultat
avait ete signalee anterieurement a propos de cestrogenes.
Des blastocystes de vison ne se sont pas developpes in vitro dans un milieu
contenant: d'autres vitamines et enzymes connus ou supposes presents dans les
liquides uterins, une serie de sources d'energie, des liquides d'uterus ou de
blastocyste de lapin, ni quand on les a transplanted dans l'uterus de lapines en
gestation depuis 3 a 4 jours.
On croit qu'il existe chez le vison un facteur inhibant la croissance du blastocyste parce que des blastocystes de lapin cessent de croitre et s'affaissent quand
on les maintient dans un milieu de culture contenant 5 % (ou davantage) de
serum de vison. Ce facteur n'est pas elimine par dialyse.
Des blastocystes de vison affaisses peuvent reprendre leur stade originel,
remplis de liquide, in vitro.
This research is supported by NSF grant number GB-4401.
The author is grateful to Mr Delbert Collicott for his co-operation in supplying, breeding,
and maintaining the mink used in these studies.
REFERENCES
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ENDERS,
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ENDERS,
{Manuscript received 19 September 1966)
J. Embryo I. exp. Morph.
Vol. 17, Part 2
PLATE
Representative mink blastocysts of some of the different ages used in this study. A, Nine days
post coitum. B, Thirteen days post coitum. C, Seventeen days post coitum. D, A 16-day
post coitum blastocyst before and (E) after 18 h of growth in vitro in culture medium containing estrone. Magnification: A, B, C, x 50; D, E, x 100.
J. C. DANIEL, JR.
facing p 302