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The role of the mucin layer in development of the rabbit blastocyst.

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The Role of the Mucin Layer in Development
of the Rabbit Blastocyst'
GILBERT S. GREENWALD
Department of A n a t o m y , University of W a s h i n g t o n ,
School of Medicine, Seattle, Washington
The rabbit is unique among eutherian tremes and marsupials. If this were true
mammals in the thick layer of mucin that the egg might be able to develop normally
coats the ovum during oviducal passage. in the complete absence of the mucin
The mucin is an acid mucopolysaccharide layer.
(Braden, '52) and is released from the
No experiments have been performed
tuba1 epithelium after ovulation (Moreaux, which directly shed light on the validity of
'13; Cohnen, '27; Westman, '30; Adams, any of these hypotheses. Recently, it was
'58; Fredricsson, '59). The secretion is found that the injection of estrogen imsynthesized by estrogen and apparently re- mediately post coiturn blocks the release
leased under the influence of progesterone of most of the mucin from the oviducal epi(Greenwald, '58). However, Flerko ('55) thelium and consequently the eggs acquire
believed that progesterone is not involved a considerably reduced mucin coating
in the discharge as judged by histological (Greenwald, '57). It was thought that the
criteria.
diminished mucin layer might account in
Several theories have been offered to part for the heavy embryonic mortality
account for the extensive deposition of that follows the administration of estrogen.
mucin around the rabbit egg. Gregory However, further studies indicated that
('30) believed that the mucin layer by re- acceleration of transport of ova during
leasing its water content was responsible the first few days of pregnancy was the
for the expansion of the rabbit blastocyst. primary factor in embryonic loss; thus the
Recent findings in relation to the problem role of the reduced imucin layer has not
of ovulation, suggest that depolymerization yet been evaluated (Greenwald, '59).
of acid mucopolysaccharides may increase
The present experiments were designed
intra-follicular osmotic pressure; this may to eliminate the accelerating effects of
account for the final spurt of growth of the estrogen noted above. This was accompreovulatory follicle (Zachariae, '59). This plished by transplanting three day old
attractive hypothesis could be equally ap- rabbit embryos from animals treated with
plicable to the marked and rapid expan- estrogen to the corriua of animals that
sion of the rabbit blastocyst.
were three days pseudopregnant. Such ova
Another theory proposed by Assheton would have a reduced amount of mucin
(1895) assigned a purely protective role and yet be subject to' a normal endocrine
to the mucin layer. The expansion of the environment during implantation.
rabbit egg is dependent most likely on
MATERIALS AND METHODS
hydrostatic pressure and the thin wall of
the blastocyst may be incapable of withA total of 139 New Zealand giant white
standing the build-up in pressure. The rigid female rabbits were used in the transtough mucin layer could act to prevent
research was supported by grant 7063 from
the rupture of the expanding blastocyst. the1 This
U. S. Public Health Service.. The human chorjonic
While both of the preceding theories gonadotropin (APL) used in thls study was furnished
the courtesy of Ayerst Laboratories, New
suggest a functional role for the mucin through
York. the estradiol cyclopentylpropionate (ECP) was
suppiied
by
the Upjohn Company, Kalamazoo, Michilayer, Hartman ('25) believed that the gan.
coating was a primitive, non-functional
2 Present address: Department of Gynecology and
University of Kansas Medical Center, Kancharacter, shared in common with mono- Obstetrics,
sas City, Kansas.
407
408
GILBERT S. GREENWALD
plantation experiments. The experimental
procedure is illustrated in figure 1. In the
control series of transfers, donor females
were mated to males of proven fertility and
the future recipients were then injected
intravenously with 100 i. u. of human
chorionic gonadotrophin (APLxx). At 70
hours post coitum the donors were killed
by the intravenous injection of an overdose
of nembutal and the oviducts were removed and flushed with 5 ml of freshly
prepared normal saline to which penicillin
(20,000 i. u. per ml) had been added. The
eggs were flushed into sterile 40 mm
square staining dishes and examined at
a magnification of 25 X with a dissecting
microscope. The diameter of each ovum
including the mucin layer was measured
with an ocular micrometer. It had been
previously determined that in this strain
of rabbits the diameter of the eggs to the
outer limit of the zona pellucida was relatively constant at 172.0 v. By subtracting
this figure from the total diameter, the
I DONORS I
diameter of the mucin layer could be determined; the thickness of the mucin layer
was then calculated by dividing by two.
Before killing the donor animal, the recipient was anesthetized with nembutal
(i.v. injection of .66 ml of standard veterinary nembutal per kg of body weight) and
the cornua exposed by a midline abdominal incision. The ova were transferred to
the uterine horns of the recipient by a
braking pipette (Holter, '43) and ordinarily, not more than 15 to 20 minutes
elapsed between the death of the donor
and the transfer of ova to the host. A week
after transplantation, the recipient was
laparotomized and the number of implantation sites counted and compared
with the number of ova that had been
transferred.
To produce eggs deficient in the mucin
layer another series of animals was injected intramuscularly immediately after
mating with 250 pg of estradiol cyclopentyl-
I
RECIPIENT
I
Induce
ovulation with
70 hours
later
70 hours PC.
Flush tubes
Measure ova
Mote:
Inject immediately
with 250 pg of
Depo- est rodiol
Laparotomy at
10 days F! C.
Fig. 1 Experimental design of the transplantation experiments. Depo-estradiol is the
trade name for estradiol cyclopentylpropionate (ECP).
MUCIN LAYER EXPERIMENTS
409
propionate (ECP). Ovulation was then induced in the recipients and the transfer
procedure outlined for the control group
was carried out. A total of 17 transfers
were made with normal ova and 40 transfers of ova with a reduced layer of mucin
(reduced mucin ova).
In an additional series of transfers with
reduced mucin or normal ova, the recipients were killed one, two or three days
after transplantation and the blastocysts
flushed from the uterus. The diameters of
the blastocysts were determined with an
ocular micrometer and compared with
blastocysts recovered from untreated animals killed on days 4, 5 or 6 post coitum
(P.c.). Twenty-five animals were used in
these experiments.
RESULTS
At 70 hours P.c., there was a marked
difference in the thickness of the mucin
layer around ova recovered from control
donors and from animals that had been
injected with ECP (figs. 2, 3). However,
ova of both groups had cleaved to the
same extent and there was no difference
in the percentage of unfertilized or abnormally developed eggs. The total diameter
of ova was usually constant within any
one rabbit in the control transfers, but
there was considerable variation from animal to animal. Ova of different animals
ranged in total diameter from 301 to 430
Fig. 2 A normal rabbit egg recovered 70 hours
post coitum. x 100.
Fig. 3 A rabbit egg recovered 70 hours post
coitum from an animal injected immediately
after mating with 250 pg of estradiol cyclopentylpropionate. Compare the thickness of the mucin
layer of both ova. ~ 1 0 0 .
p or expressed in terms of mucin thickness from 65 to 129 p.
The injection of 250 clg of ECP immediately after mating resulted in a considerable reduction of the imucin layer but did
not inhibit it completely. In 22 of 40
transfers, the ova had a mucin layer of
12 p while in the remainder it measured
up to 43 p,
The results of the transfer of ova from
the control animals are summarized in
table 1. Of a total of 148 normal eggs
transferred at three days P.c., 102 were
represented one week later by definite implantation sites (or 613.9% ). The success
of implantation had no relation to the
thickness of the mucin layer. At the time
of laparotomy, only it few embryos were
being resorbed but the close spacing of
some implantation sites (e.g. transfers
14 and 16, table 1) probably would have
resulted in further embryonic loss at later
stages in pregnancy. Only 5 females were
examined at term or shortly before delivery
(table 1, transfers 1, 5,6, 11,12). In these
animals, 23 of the original 31 implantation
sites were represented by delivered young
or living late term fetuses.
Of 332 transplanted ova with a reduced
coating of mucin, 97 eggs (or 29.2% ) implanted successfully (table 2). At 10 days
p.c. considerably more embryos were be-
410
GILBERT S . GREENWALD
TABLE 1
Results of the transfer of ova with a normal mucin thickness to recipient cornua at 3 days p.c.
Ova arranged according to thickness of mucin layer
Total
Transfer diameter
no.
egg
Mucin
thickness
No. eggs
at
transfer
No. implantation
sites
(10 days
Remarks
P.C.)
fi
P
1
2
3
4
5
6
7
8
9
10
11
430
430
409
397
397
397
387
366
366
366
366
129
129
119
113
113
113
106
97
97
97
97
10
10
10
8
11
8
4
6
8
9
11
6
9
10
6
9
5
2
4
7
4
6
12
13
14
15
16
17
344
344
344
301
301
301
86
86
86
65
65
65
10
11
9
8
6
9
5
8
3
4
6
148
102
Total
8
ing resorbed than in the control series, but
some fetuses were alive near term and
several young were delivered (table 2,
transfers 4, 5, 11, 19). Transplanted ova
with a mucin thickness of from 22 to 43 p
had a similar implantation rate (table 3).
When ova with a minimal mucin coating
(12 LI)
were transplanted only 11 out of
22 recipients became pregnant although
a number of the unsuccessful transfers
appeared to be technically perfect. Despite
this heavy loss of ova, in one transfer
(table 2, no. 19) three normal living young
were born at 34 days p.c.
The size and number of preimplantation
ova from one to three days after transfer
is shown in figure 4. At 24 hours after
transfer, the diameters of the normal ova
(now 4 days old) fell within the size range
of the control blastocysts (i.e. non-transferred group) while the reduced mucin
ova were slightly smaller. However, by 5
days, there was no significant difference in
the size of the blastocysts of the three
groups.
By 6 days p.c. there was considerable
variation in the size of the control blastocysts. For instance, in female 807 blastocysts varied from 2.3 to 3.6 mm in diameter
and in female 768 from 1.5 to 2.6 mm.
35 days p.c. - delivered 6 yg.
10 days p.c. -normal embryos
10 days p.c. - normal embryos
34 days p.c. - delivered 5 yg.
32 days p.c. - delivered 4 yg.
10 days p.c. -some adhesions
10 days p.c. - one embryo resorbing
10 days p.c. - some adhesions - 37 days - delivered 3 yg.
Killed 28 days p.c. - 5 living Yg.
10 days p.c. - one embryo at cervix
10 days p.c. - 4 embryos crowded
Poor transfer
10 days p.c. - 2 embryos crowded
Ova transferred with a normal or diminished layer of mucin had reached the same
size as the control eggs by day 6 (fig. 4).
Of 47 normal ova that were transferred,
35 (74%) were flushed three days later
from the cornua of the recipients while
out of 52 reduced mucin ova 41 (79% )
were recovered. Thus, the recovery rate
at day 6 was essentially the same for both
groups.
However, while 61.7% of the transferred control ova were large and viable
blastocysts at day 6, only 30.7% of the
blastocysts from the reduced mucin series
were normal. The other reduced mucin
ova were degenerating at day 6, apparently
after first expanding to the size of normal
day 5 ova and then collapsing (fig. 4).
It should be stressed that some of the
blastocysts developing from the reduced
mucin ova were as well developed as control embryos although most had degenerated by day 6. For example, at the time
of transfer the ova of female 843 had a
mucin thickness of only 12 p, but 5 living
blastocysts recovered from the recipient
measured 2.3 to 2.7 mm in diameter.
The recovery of blastocysts from the
recipients before implantation permitted
a comparison between the thickness of the
411
MUCIN LAYER EXPERIMENTS
TABLE 2
.
Results of the transfer o f ova w i t h a reduced mucin thickness to recipient Icornua at 3 days p.c.
Total
diameter
egg
thT$2:ss
No. eggs
at
transfer
_
No. implantation
sites
_
_
~
-_____
Remarks
(10 days
P.C.)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30-40
P
B
258
258
43
41
9
7
6
3
258
236
236
236
236
236
215
215
215
215
215
215
215
215
215
215
195
41
32
32
32
32
32
22
22
22
22
22
22
22
22
22
22
12
8
10
8
9
10
12
3
6
11
10
7
8
9
7
4
10
4
0
5
6
7
1
0
3
3
6
9
6
1
0
0
2
0
4
195
195
195
195
195
195
195
195
195
195
195
12
12
12
12
12
12
12
12
12
12
12
10
8
5
3
2
6
4
1
2
3
1
0
9
5
4
9
11
9
9
8
11
95
Total
332
10 days p.c. - adhesions - 35 days - delivered
no yg.
Perfect transfer
35 days p.c. - delivered 5 yg.
24 days p.c. - had 4 living fetuses
2 resorbing embryos at 10 days
Embryo resorbing
Perfect transfer
17 days p.c. -one resorbing
Healthy embryos
10 days p.c. - 2 resorbing - 29 days - 3 living
1resorbing - 10 days
Healthy embryos
Some eggs unfertilized?
Normal embryos
34 days - delivered
10 days p.c. - 1resorbing
3 Yg.
10 days p.c. - 2 resorbing
Healthy embryos
2 resorbing
1resorbing
1resorbing
Resorbing
See text for details
~ _ _ _
_
_
97
TABLE 3
Relationship between the mucin thickness o f the transferred reduced mucin ova
and successful implantation
~
Total
diameter
of ova at
transfer
P
258
236
215
195
195
Mucin
thickness
No. of
females
No. of
~~~~~~t
No. eggs
transferred
No.
implanted
0
24
49
75
89
95
9
19
30
39
0
24
332
97
P
43
32
22
12
12
3
5
10
11
11
Total
40
2
4
7
11
%
37.5
38.8
40.0
37.1
-
29.2
412
GILBERT S . GREENWALD
0
0
A
000
Control ova
Transferred normal ova
Transferred reduced ova
O
O
0
A
A
...HA
AAA
eeee
AA
AAA
A
00
0000
eeee
A
00
00 erne
O
0000 ee
AAAAA
e*
A
A
I
6
5
4
--
Age of ova (days post coitum)
Fig. 4 The size of individual blastocysts at 4-6 days post coitum. The asterisks designate
ova that were degenerating.
TABLE 4
Mucin thickness at 3 days p.c. and the size of individual blastocysts at 6 days
Total
diameter
of ova at
transfer
P
< 195l
195'
215l
301
38 7
430
Diameter of
recovered
6 day
blastocysts
Mucin
thickness
mm
P
< 12
3.3, 1.4
2.3,2.7,2.4,2.7,2.5
3.0, 2.1,2.5,2.2,3.0,2.8, 1.9
2.0,2.0,2.2,2.0
2.6,2.6,2.9,2.2,3.0,3.0,3.0,3.i
2.3,2.9, 3.5,3.4,3.4,4.0,2.0,3.6
12
22
65
106
129
'Donor animals had been injected with
ECP.
mucin layer of day three tuba1 ova and the
ultimate size reached by the blastocysts.
Six representative transfers involving ova
ranging from 195 to 430 in total diameter are listed in table 4. The 5 largest
blastocysts recovered corresponded to ova
that were 430 in diameter at the time
of transfer. With these exceptions, there
appears to be no significant positive corre-
lation between initial thickness of the mucin layer and the size of the blastocysts.
DISCUSSION
It is apparent that reduction of the
mucin layer to a thickness of only 12 p is
not incompatible with normal implantation
and development of the rabbit egg. However, the rate of successful implantation
was sharply reduced (tables 1, 2).
MUCIN LAYER EXPERIMENTS
After the present research was completed, Adams reported some preliminary
experiments on the role of the mucin layer
in development of the rabbit blastocyst
('60). Two-celled ova were recovered and
cultured in vitro for 24 hours; 52 moruIae
with a mucin layer of 10 M or less were
then transferred to 7 recipients. At laparotomy at 10 days P.c., 44% of the eggs
had implanted successfully in 5 recipients.
These results are in substantial agreement
with the present findings.
The reduced mucin ova in the present
experiments were produced by injecting
rabbits immediately after mating with
250 ug of estradiol cyclopentylpropionate
(ECP). Estrogens have a deleterious effect
on cleaving sea urchin eggs by arresting
mitoses (Agrell, '54) but there is no evidence that exogenous estrogens directly
influence the development of mammalian
ova. In the present experiments, ECP did
not interfere with the normal cleavage pattern; the ova with a reduced mucin layer
were identical at three days p.c. with normal untreated ova (figs. 2, 3). Similarly,
rabbit ova cultured in vitro with estrone or
estradiol go through cleavage stages at the
normal rate and diazo compounds coupled
with estrone fail to penetrate and vitally
stain such ova (Pincus and Kirsch, '36).
The injection of 5 mg of stilbestrol at 5
days p.c. also does not alter the mitotic
rate in the extra-embryonic layer of the
rabbit blastocyst (Moog and Lutwak-Mann,
'58).
While there are no indications that rabbit ova are directly affected by estrogen
and while there are no visible effects on
the morula stage, a deleterious effect on
the blastocysts at the time of the abrupt
morphological and biochemical reorganization of the developing blastocyst (Brachet,
'60) cannot be excluded. It is possible that
the greater mortality of the reduced mucin
ova is due in part to their development in
an environment dominated by estrogen.
It is apparent from figure 4 and table 4
that the thickness of the mucin layer at
day three does not correlate with the ultimate size reached by the blastocyst. By day
6 P.c., some reduced mucin ova had expanded to the same size as normal blastocysts, This appears to rule out Gregory's
413
theory that the muciri layer is necessary
for expansion of the blastocyst ('30).
The degeneration of blastocysts developing from reduced mucin ova might indicate a protective role of the mucin layer
in accordance with Assheton's hypothesis
(1895), but a number of the reduced mucin ova grew into large and viable blastocysts at day 6. Thus, the results of the
transplantation experiments do not provide clear-cut evidence for any specific
protective role for the mucin layer.
However, the most critical experiment
has not yet been performed. Until ova
completely devoid of any trace of mucin
are transplanted, the possibility still exists
that the blastocyst may require only a thin
coating for its normal differentiation. Preliminary attempts to produce such mucinless ova have so far been unsuccessful.
After increasing the single post-copulatory
injection of ECP from 250 to 500 clg, the
minimal thickness of mucin was unchanged at 12 CI (Greenwald, unpublished).
This suggests that minimal amounts of
mucin may always be covering the surface
of the tubal epithelium and that exogenous
estrogens prevent only the release of the
stored intra-epithelial content.
A n alternative explanation for the failure to inhibit mucin release completely
with estrogens could be the rapid release
of progesterone within 100 minutes after
coitus (Forbes, '53). Progesterone is responsible for the normal secretion of mucin and exogenous estrogens prevent the
discharge by competing with proeesterone
(Greenwald, '58). The initial rapid release
of progesterone could lead to a limited discharge of mucin before effective inhibitory
levels of circulating estrogen are reached.
To test this possibility, rabbits were injected two or three days before ovulation
with 250 pg of ECP and ovulation was
then induced with gonadotrophins. However, after this treatment the recovered
tubal ova were still invested with a thin
mucin layer.
The mucin layer can be reduced markedly by injecting recently mated rabbits
with estrogens (fig. 3) or by removing twocelled ova from the oviduct and culturing
them in in vitro (Adams, '60). It is possible that the thin muciii layer surrounding
such ova could be dissolved with proteo-
414
GILBERT S. GREENWALD
lytic enzymes (Braden, ’52) without affecting the egg itself. The role, if any, of the
rnucin layer cannot be established with
certainty until ova without any trace of the
coating are transferred to the cornua of
pseudopregnant recipients.
SUMMARY
These experiments were designed to determine whether or not the mucin layer
is essential for development and implantation of the rabbit blastocyst. Usually, the
normal three day old morula is invested by
a mucin layer varying in thickness from
65 to 129 w. In previous experiments this
layer was greatly reduced after estrogen
was injected into rabbits immediately after
coitus. In order to circumvent the effect
of estrogen on transport of ova and development of the endometrium, ova with a
layer of mucin as thin as 12 p were produced by injecting does immediately post
coitum with 250 ug of estradiol cyclopentylpropionate. The ova were transplanted
at three days p.c. to the cornua of pseudopregnant recipients and the success of implant ation determined.
Of a total of 332 ova transferred with a
reduced layer of mucin, 97 (or 29.2%) implanted one week later. When 148 control
ova were transferred similarly, 68.9% implanted successfully. Since reduction of
the mucin layer was the only variable it
appears that lack of sufficient mucin interfered with implantation. However, in one
instance normal living young were delivered at term that had developed from eggs
with an envelope of mucin as thin as 12 I-I.
Furthermore, 6 day blastocysts recovered
during the preimplantation period and derived from ova similarly deficient in the
mucin layer were found to have expanded
to the same size as normal embryos. By
day 6 only 31% of such ova were large
and viable blastocysts; the remainder were
degenerating after developing to the same
size as normal 5 day eggs.
These experiments indicate that normal
expansion and implantation of the rabbit
blastocyst sometimes occurs despite a
marked reduction in the thickness of the
mucin layer. The mucin layer may not be
essential, therefore, for normal development of the rabbit egg. However, it re-
mains to be established whether ova completely devoid of any coating of mucin can
undergo normal development.
LITERATURE CITED
Adams, C. E. 1958 Egg development i n the
rabbit: the influence of post-coital ligation of
the uterine tube and of ovariectomy. J. Endocrin., 16: 283-293.
1960 Development of the rabbit eggs
with special reference to the mucin layer. Advance Abstracts of the First Intl. Congress of
Endocrinol., Abstract No. 345, p. 687, Copenhagen.
Agrell, I. 1954 Oestradiol and testosterone propionate as mitotic inhibitors during embryogenesis. Nature, 173: 172.
Assheton, R. 1895 A reinvestigation into the
early stages of development of the rabbit.
Quart. J. Micr. Sci., 37: 113-164.
Brachet, J. 1960 The biochemistry of development. Pergamon Press, New York.
Braden, A. W. H. 1952 Properties of the membranes of rat and rabbit eggs. Aust. J. Sci. Res.
Ser. B. BioI. Sci., 5: 460471.
Cohnen, K. 1927 Uber den Mechanismus der
Eiwanderung durch den Eileiter mit besonderer
Beriicksichtigung der zyklischen Veranderungen
am Eileiterepithel des Kaninchens. Ztschr. Mikroskop. Anat. Forsch., l l : 4 7 s 4 9 2 .
Flerko, B. 1955 Die Epithelien des Eileiters und
ihre hormonalen Reaktionen. Ibid., 61: 99-118.
Forbes, T. R. 1953 Pre-ovulatory progesterone
in the peripheral blood of the rabbit. Endocrinology, 53: 79-87.
Fredricsson, B. 1959 Studies on the morphology and histochemistry of the fallopian tube
epithelium. Acta Anat. Supp. 37, 38: 1-23.
Greenwald, G. S. 1957 Interruption of pregnancy in the rabbit by the administration of
estrogen. J. Exp. Zool., 135: 461-482.
1958 Endocrine regulation of the secretion of mucin in the tuba1 epithelium of the
rabbit. Anat. Rec., 130: 477496.
1959 The comparative effectiveness of
estrogens in interrupting pregnancy in the
rabbit. Fertility and Sterility, 10: 155-161.
Gregory, P. W. 1930 The early embryology of
the rabbit. Carnegie Inst. Wash. Pub. 407, Contrib. to Embryol., 21: 141-168.
Hartman, C. G. 1925 On some characters of
taxonomic value appertaining to the egg and
the ovary of rabbits. J. Mammalogy, 6: 114121.
Hay, M., C. E. Adams and C. Lutwak-Mann 1959
The effect of certain agents upon the early rabbit embryo. J. Endocrinol., 20: ii-iii.
Holter, H. 1943 Technique of the Cartesian
diver. Compt. rend. Lab. Carlsberg, SBr. Chim.,
24: 399478.
Moog, F., and C. Lutwak-Mann 1958 Observations on rabbit blastocysts prepared as flat
mounts. J. Embryol. Exp. Morph., 6: 57-67.
Moreaux, R. 1913 Recherches sur la morphologie et la fonction glandulaire de 1’6pith6lium
MUCIN LAYER EXPERIMENTS
de la trompe uterine chez les mammiferes.
Arch. d'Anat. Micr., 14: 515-576.
Pincus, G., and R. E. Kirsch 1936 The sterility
in rabbits produced by injections of oestrone
and related compounds. Am. J. Physiol., 11 5:
219-228.
415
Westman, A. 1930 Studies of the function of
the mucous membrane of the uterine tube.
Acta Obstet. Gynec. Scand., 10: 288-298.
Zachariae, F. 1959 Acid mucopolysaccharides
in the female genital system and their role in
the mechanism of ovulation. Doctoral Thesis.
Periodica. Copenhagen, 1-63.
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