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Endometrial and embryonic enzymes in relation to implantation of the rabbit blastocyst.

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Endometrial and Embryonic Enzymes in Relation to
Implantation of the Rabbit Blastocyst
E. s. E. HAFEZ AND I. G . WHITE
Department of Veterinury Physiology, University of Sydney,
Sydney, N. S. W. Australia
ABSTRACT
The activity of alkaline phosphatase, acid phosphatase, plutamic-oxdacetic transaminase (GOT),lactic dehydrogenase (LDH), amylase, succinic dehydrogenase (SDH), glucose-&phosphate dehydrogenase (GD) has been determined in the
rabbit endometrium at estrus, in pseudopregnancy, and at days 5-10 of pregnancy.
Enzyme analyses were also made on the placenta and embryo during early pregnancy.
The most striking changes were increases in the SDH and GDH levels of the endometrium and a decrease in amylase when the does became pregnant or pseudopregnant.
Amylase did not rise significantly from its lower level between five and ten days of
pregnancy; SDH and GDH activity, however, fell away after reaching a maximum
prior to implantation. At implantation there was some evidence of an increase in
LDH and phosphatase activity. All enzymes had lower activities following implantation (10 days pregnancy). The endometrial enzyme activities of prepubertal does were
usually similar to those of adult females in estrus. However, alkaline phosphatase
activity of the prepubertal endometrium was particularly low.
The activity of enzymes in the placental areas were, in general, similar to those
found in the interplacental areas of the endometrium. However, from 7-10 days of
pregnancy the activity of SDH was lower in the placental area than in the interplacental areas; whereas, amylase was higher on the eighth and ninth days of pregnancy.
Changes in the activities of the phosphatases, GOT and SDH occurred in the blastocyst
and trophoblast on eight to ten days of pregnancy. Enzyme activities in blastocoelic
fluid were much less than in the trophoblast with the exception of amylase which was
higher.
The female reproductive tract possesses
a wide and characteristic distribution of
enzymes with varying activity depending
on the endocrine condition and the stage
of the reproductive cycle (cf. Hafez, '64).
In the rabbit endometrium, dipeptidase activity increases rapidly from estrus to
fourth day of pseudopregnancy. This is
followed by four days of a much slower
rise, four days of a maintained plateau
and five days of a steady decline until a
level slightly lower than that present during estrus is reached (Albers, Bedford and
Chang, '61). The carbonic anhydrase activity of the rabbit endometrium has also been
shown to increase during early or pseudopregnancy (Lutwak-Mann and Laser, '54;
Lutwak-Mann, '55) and Boving ('62) has
suggested that it causes local alkalinity
which makes both the membranes and the
trophoblast sticky and thus promotes implantation. According to Delgado and Fridhandler ('64) the lactic dehydrogenase
activity of the rabbit endometrium, particularly in the interplacental region, is decreased in early pregnancy whilst malic
ANAT.
REC..159: 273-2230.
-
dehydrogenase and glutamic oxalacetic
transaminase activity is increased from
five to seven days of pregnancy.
In the rat, uterine p-glucuronidase (Prahald, '62) and endometrial peptidase have
been shown to increase at implantation
(Albers et al., '61). The occurrence of
proteolytic enzymes in the uterus capable
of digesting collagens as well as muscle
protein (Morione and Swifter, '62; Woessner and Brewer, '63) may also be important
for implantation.
This report describes fluctuations in the
activity of a number of enzymes - alkaline
phosphatase, acid phosphatase, glutamicoxalacetic transaminase (GOT), lactic dehydrogenase (LDH), amylase, succinic dehydrogenase (SDH) , glucose-&phosphate
dehydrogenase (GDH) - in the rabbit
~~
XPresent address: Department of Animal Sciences,
Washington State Univmsity, Pullman Washington
U. S. A. College of Agriculture Scientdc Paper 2958:
Project 1695.
2 We wish to thank Professor C. W. Emmens for
providing laborato
facilities and for his. interest
and advice, and x e U. S. National Insbtutes of
Child Health and Human Development for a Special
Fellowship (IF3 HD-24. 411-01) while one of us
(E.S.E.H.) was on sabbatical leave, 1965/66.
2 73
274
E.
S.
E. IIAFEZ AND I. G . WHITE
endometrium and embryo during early
pregnancy.
MATERIAL AND iMETHODS
1. Experimental animals
Rabbits of mixed breeds, mostly New
Zealand White, were obtained from the
University of Sydney animal house at Castle Hill. All does were primiparous, aged
7-10 months, except the prepubertal (nonpregnant) group which were 3-4 months
old and the aged (pregnant) group which
were 2-3 years old and had over five
previous pregnancies. Does were made
pseudopregnant by intravenous injection
20 I.U. of Human Chorionic Gonadotropin
(HCG) (Schering) and pregnant by simultaneous insemination with 0.1 ml of pooled
semen freshly collected with an artificial
vagina (White, '55).
2. Preparation of tissues
The does were killed at the reproductive
stages indicated in table 2 and the uteri
removed immediately and immersed in
crushed ice. All subsequent manipulation
of the tissue was performed at 4°C. Blastocysts 5 to 6 days of pregnancy (post coitum)
were flushed from the uterine horns with
calcium-free Krebs-Ringer buffered to pH
= 7.0 with Tris (122 mM NaC1, 5 mM
KCl, 1 mM MgS04, 25 mM Tris) and the
healthy ones (as judged by stereoscopic
examination) transferred to a homogenizing tube. Seven-day old blastocysts were
removed after making three incisions i n
the uterine wall near each blastocyst; a
strip of uterine wall was then peeled off
using fine forceps, releasing each blastocyst.
The maternal placenta, trophoblast and
blastocoelic fluid were collected from eight,
nine and ten days of pregnancy. After
three incisions were made in the uterus
2 mm from the conceptus, the uterine wall
was teased around the conceptus using
two fine forceps. The blastocoelic fluid
(ca. 0.5 ml/conceptus) was collected in a
small pre-cooled petri dish. The antimesometrial side of the uterus was turned inside out over a finger and the trophoblast
scraped from the uterine wall using the
edge of fine forceps. The uterine wall surrounding the blastocyst was dissected to
determine if any trophoblastic tissue remained after cleaning with tissue paper or
cheese cloth. The uterine horn was then
stretched on a piece of filter paper and
opened at the antimesometrial side. The
exposed endometrium was blotted with filter paper to remove any traces of the flushing fluid or the endometrial secretions.
Tissues from the placental fold and the
interconceptus endometrial areas were dissected using fine forceps.
3. Enzyme analyses
Samples of tissue ( 0 . 5 to 1 gm) were
homogenized in ten parts of calcium-free
Krebs-Ringer-Tris except in the case of
blastocysts when two parts were used. A
teflon Potter-Elvehjem homogenizer was
found to be satisfactory and after filtering
through cheese cloth enzymes were assayed either directly or after appropriate
dilution in calcium-free Krebs-Tris (table 1)
by assay procedures which, except for succinic dehydrogenase, are set out in Bergmeyer ('63).
TABLE 1
The dilution of rabbit tissue homogenates and fluid for enzyme analyses
One volume of the homogenate (see Materials and Methods for preparation) or fluid was diluted
to the following volumes with calcium free Krebs-Ringer-Tris. N.D. indicates that the homogenate or
fluid was not diluted.
Alkaline
Homogenate
Endometrium
Placenta
Blastocyst
Trophoblast
Fluid
&%&e
20
20
10
20
2
Acid
phosphatase
10
10
10
10
5
GOT
10
10
5
10
N.D.
Lactic
dehydrogenase
10
10
10
10
N.D.
Amylase
N.D.
2
10
2
20
Succinic
dehydrogenase
2
N.D.
N.D.
N.D.
N.D.
Glucose-6phosphate
dehydrogrrnnse
_ - - ---
20
20
N.D.
10
N.D.
ENZYME ACTIVITIES DURING IMPLANTATION
pNitrophenylphosphate was used as the
substrate for determining acid and alkaline phosphatase activity (Bessey, Lowry
and Brock, '46; Andersch and Szcypinski,
'47). One phosphatase unit is the amount
of enzyme contained in 1,000 ml of homogenate which liberates 1 m.mole (139.11
mg) p-nitrophenyl at 37°C.
Glutamic-oxalacetic transaminase (GOT)
activity was measured by determining the
rate of formation of oxalacetate colorimetrically as the 2,4-&nitrophenylhydrazine
(Reitman and Frankel, '56). The optical
density at 546 mv was converted to GOT
units from the table in Bergmeyer ('63).
A GOT unit is the amount of transaminase
in 1 ml of homogenate which decreases
the optical density of DPNH at 340 mu by
0.001 in one minute, in a 3 ml assay mixture at 25°C (La Due, Wroblewski and
Karmen, '54 ) .
Lactic dehydrogenase (LDH) activity
was measured by the decrease of optical
density at 340 my due to oxidation of
DPNH in the prcsence of pyruvatc. A unit
is the amount of LDH which changes the
optical density of DPNH at 340 my by
0.001 in one minute; in a 3 ml assay mixture at 24-27°C (Wroblewski and La Due,
'55).
Amylase was estimated by the change in
iodine color of an amylase solution (Street
and Close, '56) using one tenth of the
volumes set out in Bergmeyer ('63) for the
micro method. Under our conditions one
Street-Close unit would be contained in
10 ml of homogenate when 0.1 ml hydrolysed 0.2 mg of amylase in 15 minutes at
pH 7.0 and 37°C.
Succinic dehydrogenase (SDH) was determined by the reduction of neotetrazolium chloride with sodium succinate as
substrate (Eckstein, Kahan and Borut, '57;
Sobel and Eckstein, '59). The succinic dehydrogenase activity of the homogenate
was expressed as micrograms of neotetrazolium reduced during 20 minutes at 37°C.
Spectrophotometric determination of the
rate of TPNH formation was used for the
assay of glucose-6-phosphate dehydrogenase (GDH) (Warburg, Christian and
Griese, '35). One unit of activity is the
amount of enzyme in 1 ml of sample which
at 25°C in a 3 ml assay mixture changes
the optical density of TPNH at 340 my by
275
0.001 in one minute (La Due, Wroblewski
and Karmen, '54).
The protein concentration of homogenates was determined by the biuret method
(Wales, Scott and White, '61) and dry
weight determined after three hours at
105°C.
Analyses of variance were done using
the SILLIAC electronic computer and the
interaction mean square used to calculate
standard errors of the difference between
means, for t-tests.
RESULTS
1. Endometrium
Table 2 shows the activity of alkaline
phosphatase, acid phosphatase, GOT, LDH,
amylase, SDH and GDH per mg of endometrial protein in the rabbit at estrus, in
pseudopregnancy and at days 5 to 10 of
pregnancy. Samples from prepubertal (nonpregnant) does are also included for comparison.
The most striking changes are the significant increases in SDH and GDH and
the decrease in amylase when the doe becomes pregnant or pseudopregnant. Implantation in the rabbit occurs at nine days
of pregnancy as judged from histological
evidence and studies on the vascular architecture of the endometrium in relation to
the blastocyst. Prior to this time, the endometrium undergoes morphological changes
and proliferation (cf. Hafez and Tsutsumi,
' 6 6 ) . Table 2 shows the variations in enzyme activity that occur during this stage
of development. Amylase did not rise significantly from its lower level between 5
and 10 days of pregnancy. SDH and GDH
activity, however, fell away after reaching
a maximum prior to implantation. At implantation there is the suggestion of an
increase in the activity of LDH and the
two phosphatases. All the enzymes assayed had lower activities following implantation (10 days of pregnancy) than
before.
The endometrial enzyme activities of
old-multiparous does compared to youngprimiparous does, seven days of pregnancy,
were similar except for the higher activity
of alkaline phosphatase (733 and 550 X
lo+ units per mg of protein, respectively)
and lower activity of SDH (498 and 971
276
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E. HAFEZ AND I. G. WHITE
X lo-* units per mg of protein, respectively) in the older animals (table 2).
In prepubertal does, most enzyme activities were similar to adults in estrus, and
with the exception of amylase, lower than
in pregnant does. The alkaline phosphatase level of the prepubertal endometrium
was particularly low.
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2. Placental areas
The activity of the enzymes in the placenta 7 to 10 days of pregnancy are shown
in table 3. There was a statistically significant increase in alkaline phosphatase
and amylase at or near implantation, i.e.
8-9 days of pregnancy. No large differences were found between multiparous and
primiparous does seven days of pregnancy.
Values for SDH were higher than for the
comparable stages of development. As
with the endometrium, the activity of all
enzymes was lower on day 10 than 9.
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3. Blastocyst and t r q h o b l a s t
Alkaline and acid phosphatase were detected in one pooled sample of blastocyst
as early as five days of pregnancy, the
values being 10 and 8 X
units mg
protein respectively. The small amount of
tissue available, however, did not permit
detection of the other enzymes at this stage
of development. The activity of acid phosphatase, GOT and SDH in the blastocysts
was significantly higher on the eighth day
compared to the sixth. The results also
suggest that the seven-day blastocysts from
multiparous animals have higher GOT and
amylase activity and lower LDH activity
than comparable blastocysts from primiparous animals. Insufficient material was
available, however, to prove this point
statistically.
In the trophoblast, the activity of the
two phosphatases and possibly also SDH
decreased gradually from eight to ten days
of pregnancy. The activity of GOT and
probably also LDH increased markedly at
implantation (9 days of pregnancy) and
then declined.
The enzyme activities in the blastocoelic
fluid were generally 10 to 100-fold less
than in the trophoblast; the only exception
was amylase, which was 2 to 5-fold higher.
The activity of LDH increased from eight
days to ten days of pregnancy, whereas the
ENZYME ACTIVITIES DURING IMPLANTATION
277
activity of alkaline phosphatase and amylase showed a tendency to decline. SDH and
GDH could not be detected with any certainty.
DISCUSSION
-%
*
-%
*
During the early stages of development,
the blastocyst must become attached to
the endometrium but little is known about
the changes of the enzyme in the endometrium and blastocyst that allow this to
occur. The main episodes in nidation are
the preparation of a uterine zone of receptiveness in which the blastocyst may become embedded and the active penetration
of that zone by the blastocyst or the attachments of the blastocyst by the uterine
tissue.
The present results suggest that under
the influence of progesterone the SDH and
GDH activity of the rabbit endometrium
increases and the amylase levels fall. Telfer and Hisaw ('57) report a similar
progestational response in endometrial succinoxidase, an enzyme which plays such
an important part in the tricaboxylic acid
cycle. GDH is the enzyme giving access to
the pentose shunt and an increase in both
oxidative pathways may be a prerequisite
for implantation.
Our results could be interpreted to mean
that at implantation the blastocyst has a
differential effect on the enzyme levels of
the interplacental and placental areas.
From seven to ten days of pregnancy, the
activity of SDH was lower in the placental
area than in the interplacental areas,
whereas amylase was higher on days 8 and
9 of pregnancy. There were slight variations, but no significant trends between the
activity of the other enzyme in these two
areas. A decline, however, occurred in the
activity of all the enzymes in both areas
following implantation.
These differences in enzyme levels, as
well as differences in the placental tissues
at different stages of pregnancy, may, of
course, be partly due to differences in the
degree of the vascularity of the placenta
and the amount of blood engorged in the
tissues. The vascularity of the placental
tissues increases markedly at nine days of
pregnancy (Hafez and Tsutsumi, '66).
Individual differences in enzyme activity
at the same stage of pregnancy may be
278
E. S. E. HAFEZ AND I. G . WHITE
related to differences in the degree of
physiological maturity of the uterus.
The determinations of LDH and SDH in
the blastocyst and trophoblast are consistent with Fridhandler's ('61) finding that
after six days the rabbit blastocyst is capable of metabolizing glucose via the Embden-Meyerhof pathway and the tricarboxylic acid cycle. The switch from the
hexose monophosphate oxidation pathway
at this stage of blastocyst development is
presumably not due to loss of GDH activity
since it could still be detected in the blastocyst and trophoblast on the tenth day after
mating.
The changes in the activities of acid
phosphatase, GOT and SDH in the blastocyst and trophoblast on days 8-10 of pregnancy may in some way be related to the
implantation process. Lutwak-Mann ('59)
has shown that the concentration of potassium, bicarbonate, protein and glucose in
the blastocyst fluid of the rabbit change at
implantation in the direction of the concentration in the maternal blood. This
seems unIikely to be the explanation for
the increase in the LDH of the blastocyst
fluid, however, since the concentration is
about three times that of rabbit plasma on
day 10 of pregnancy.
It is known that the giant cells of the
trophoblast perform several functions including invasiveness and synthesis of luteotrophic and/or steroid hormones. The
histological appearances at the margin of
the growing trophoblastic shell of the human placenta in the early months of gestation suggest that the cells and matrix of the
decidua are attacked and slowly destroyed
by the action of the advancing growing
cytotrophoblast (Wislocki and Padykula,
'61). In fact, fragmentation of collagen
fibers in the vicinity of the trophoblast has
been notcd in the placenta of rodents
(Wislocki, Dean and Dempsey, '46).
The previous history of the female may
have an effect on the enzyme distribution.
Thus the endometrium of aged animals
had a lower activity of succinic dehydrogenase and possibly higher activity of alkaline phosphatase than that of primiparous
animals which may be related to the effect
of aging on the rate of placental development and rate of implantation.
**
cy*
d
*
Q
**
4-
*
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ENZYME ACTIVITIES I JRING IMPLANTATION
279
tribution and hormonal dependence. J. EndoThe enzymes present in prepubertal and
crinol., 13: 26-38.
mature (non-pregnant) endometrium were
1959 Biochemical approach to the study
also found to differ in some respects. Thus
of ovum implantation in the rabbit. Memoirs
SOC.Endocrinol., 6: 35-49.
the prepubertal does had a low activity Qf
C., and H. Laser 1954 Bicarboalkaline phosphatase while the levels of Lutwak-Mann,
nate content of the blastocyst fluid and carbonic
LDH, amylase, SDH and GDH tended to be
anhydrase in the pregnant rabbit uterus. Nahigher. The significance of these findings
ture, 173: 268-269.
is not clear, but may be correlated with the Morione, T., and S. Swifter 1962 Alteration in
the collagen content of the human uterus durmaturation of the female reproductive
ing pregnancy and postpartum involution. J.
tract.
Exp. Med., 115: 357-365.
LITERATURE CITED
Albers, H. J., J. M. Bedford and M. C. Chang
1961 Uterine peptidase activity in the rat and
rabbit during pseudoprcgnancy. Amer. J.
Physiol., 201: 554-556.
Andersch, M. A., and A. J. Szcypinski 1947 The
use of p-nitrophenylphosphate as substrate in
determination of serum acid phosphatase. Amer.
J. Clin. Pathol., 17: 571-574.
Bergmeyer, H. 0. 1963 Methods of enzymic
analysis. Academic Press, New York, pp. 559.
Bessey, 0. A,, 0. H. Lowry and M. J. Brock 1946
A method for the rapid determination of alkaline phosphatase with five cubic millimetres of
serum. J. Biol. Chem., 164: 321-329.
Boving, G. G. 1962
Chap. VII, Mechanisms
Concerned with Conception (edited by C. Hartm a n ) , Pergamon Press, London: 321-396.
Delgado, R., and L. Fridhandler 1964 Enzyme
changes in the rabbit uterus in early pregnancy.
Exp. Cell. Res., 34: 45-53.
Eckstein, B., D. Kahan and A. Borut 1957 A
modification of the tetrazolium reaction method
for quantitative determination of dchydrogenase activity in tissues (preliminary note).
Bull. Res. Counc. Israel, Sec. E, 6E: 189-192.
Fridhandler, L. 1961 Pathways of glucose
metabolism in fertilized rabbit ova a t various
pre-implantation stages. Exp. Cell. Res., 303:
303-316.
Hafez, E. S. E. 1964 Uterine and placental
enzymes. Acta Endocrinologica, 46: 217-229.
Hafez, E. S. E., and E. S. Tsutsumi 1966 Endometrial vascularity during pseudopregnancy in
the rabbit. J. Morph., 118: 43-56.
La Due, T. S., F. Wroblewski and A. Karmen
1954 Serum glutamic oxalacetic transaminase
activity i n human acute transmural myocardial
infection. Science, 120: 497-499.
Lutwak-Mann, C. 1955 Carbonic anhydrase in
the female reproductive tract. Occurrence, dis-
Prahald, K. V. 1962 A study of the r a t pglucuronidase prior to implantation of the
ovum. Acta Endocrin., 39: 407-410.
Reitman, S., and S. Frankel 1956 A colorimetric method for the determination of serum
glutamic oxalacetic and glutamic pyruvic transaminases. Amer. J. Clin. Pathology, 28: 56-63.
Sobel, H., and B. Eckstein 1959 Succinic dehydrogenase activity in brain tissue of rats
after ovariectomy and steroid administration.
Nature (Lond.), 183: 54-55.
Street, H. V., and J. R. Close 1956 Determination of amylase activity in biological fluids.
Clin. Chim. Acta, I: 256-268.
Tclfer, M. A., and F. L. Hisaw 1957 Biochemical resuonses of the rabbit endometrium and
myome&ium to oestradiol and progesterone.
Acta Endocrin. (Kbh.), 25: 390404.
Wales, R. G.,T. W. Scott and I. G. White 1961
Biuret reactive materials in semen. Aust. J.
Exp. Biol. Med. Sci., 39: 455-462.
Warburg, O., W. Christian and A. Griese 1935
Wasserstoffubertragendes Co-Ferment seine zusanimensetzung und Wirkingsweisi. Biochem.
Zeits., 282: 157-205.
White, I. G. 1955 The collection of rabbit
semen, Aust. J. Biol. Sci., 33: 367-370.
Wislocki, G. B., H. W. Deane and E. W. Dempsey
1916 The histochemistry of the rodent's placenta. Am. J. Anat., 78: 281-345.
Wislocki, G. B., and H. A. Padykula 1961 Histochemistry and electron microscopy of thc placenta. Chap. 15. In: Sex and I n t e r n d Secretions, (ed. by W. C. Young). Bailliere, Tindall
& Cox, Ltd., London, 2: 883-957.
Woessner, J. F., Jr., and T. H. Brewer 1963
Formation and breakdown of collagen and
elastin in the human uterus during pregnancy
and postpartum involution. Biochem. J., 89:
75-82.
Wroblewski, F., and J. S. La Due 1955 Lactic
dehydrogenase activity in blood. Proc. SOC.
exp. Biol. Med., 90: 210-213.
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