BioSMART ISSN: 1411-321X

Volume 2, Nomor 1 April 2000

Halaman: 1 - 6

Regulation of Glycogenolysis in the Uterus of the Mouse during Post-

implantation Pregnancy 1: Hormonal Control

SUTARNO
Jurusan Biologi FMIPA UNS Surakarta

ABSTRACT

The present study investigated the pattern of glycogen deposition and the regulation of glycogenolysis in the liver and uterus of the
mouse during peri- and post-implantation pregnancy.The results showed that through days 3 to 13 of pregnancy, uterine glycogen
both in the endometrial tissue and the whole uterus changed significantly with advancing pregnancy. Thus, glycogen concentration
in endometrium increased significantly through days 3 and 9, and then slightly decreased through day 13. Similarly, glycogen
concentration in the whole uterus increased through days 3 to 9 but remained unchanged through days 9 to 13 of pregnancy.
However, endometrial glycogen concentration were higher than in the whole uterus, suggesting that glycogen was deposited
mainly in the decidualized endometrium during peri- and post-implantation pregnancy. The total content of glycogen per uterus
underwent more than a 50-fold increase from an initial value about 0.1193 mmol of glycosyl units/uterus to approximately 6.0321
mmol of glycosyl units/uterus on day 13. Treatment of day 9-pregnant mice with either ethanol (3.0 g/kg body weight),
epinephrine (1mg/kg body weight) or glucagon (1 and 10 µg/mouse) decreased (P<0.001) the concentration of liver glycogen 1h
after treatment. However, only glucagon at the high dose (10 µg/mouse) slightly decreased the uterine concentration of glycogen
(P<0.05). Whether or not the hormones affect phosphorylase activity will be determined in the next publication.

Key words: glycogenolysis, uterus, mouse, pregnancy, hormonal control

INTRODUCTION liver, where it serves as a buffer to maintain a

Peri-implantation stages of pregnancy represent
a most crucial period for the developing embryo.
While advances in understanding the events of this
period and their application to fertility control, both
in humans and commercially valuable animals have
been made in recent years, the physiology and
biochemistry of the peri-implantation stages of
pregnancy are still incomplete. One of the
important metabolic aspects, glycogenolysis, that
occurs in the uterus during post-implantation period
also remains unclear and need to be investigated
more extensively.
Immediately after implantation of blastocyst, the
endometrium undergoes decidualization, a process
involving the transformation of endometrial stromal
fibroblasts to form decidual cells (De Feo, 1967).
These cells are different from the normal fibroblast
in many aspects, and their presence can be detected
from the external appearance of the uterus, where
they cause swellings at the site of implanting
blastocyst. Each swelling is an organized structure,
the decidua, in the centre of which is the
implantation chamber.
Glycogen, a polymeric form of glucose stored in
animal cells, which can be degraded on demand. In
most tissue, including muscle, the role of glycogen
is as a glycolytic fuel that provides energy locally
when glucose or oxygen becomes scarce. In the
constant level of blood glucose, glycogen is broken
down to release glucose between meals. In contrast
when glucose supply is abundance, the liver can
convert glucose into glycogen. Muscle glycogen
serves as an important source of energy for muscle
contraction, although a major portion of the energy
supply of muscle comes from the breakdown of
fatty acids.
In uterus, the importance of uterine glycogen
has been suggested as an energy store for the
metabolic demands of egg implantation (Walaas,
1952) and it also act as an important energy source
for both embryonic development (Demers et al.,
1972) and parturition (Chew and Rinard, 1979).
These functions are supported by the evidence that
glycogen accumulation appears cyclically during
oestrus cycle.
The objectives of this experiment were:
1. To investigate the pattern of glycogen
deposition in the uterus during peri- and post-
implantation pregnancy, and
2. To investigate the role of hormones on glyco-
genolysis both in liver and uterus of the mouse
during peri-and post-implantation pregnancy.
Whether or not the hormones affect
phosphorylase activity will be determined in the
next publication.
© 2000 Jurusan Biologi FMIPA UNS Surakarta
2 SUTARNO – Glycogenolysis 1. Hormonal Control
MATERIALS AND METHODS
Experimental Animals
The experimental animals used in all researches
were outbreed Quackenbush (QS) strain mice aged
between 6 to 10 weeks. They were housed in white
plastic cages under controlled environmental
conditions (constant temperature of 22oC, with
unlimited access to fresh tap eater and food).
Pregnancy was brought about by pairing females
with fertile males of the same strain. The females
were examined for copulation plugs each morning,
and the day of finding a plug was designated as day
1 or the first day of pregnancy. Some animals were
killed by cervical dislocation between 9.00 and
10.30 h on days 3, 5, 7, 9, 11 and 13 of pregnancy
without receiving any further treatment. These
animals were used to establish the normal patterns
of glycogen deposition in the uterus during the peri-
and post-implantation stages of pregnancy. In other
groups, mice were treated with either saline,
ethanol, epinephrine or glucagon on day 9 of
pregnancy to asses the role of these agents in the
regulation of liver and uterine glycogenolysis.
Treatments and procedures
Four groups of day-9 pregnant QS mice were
treated with ethanol, epinephrine or glucagon (or
saline as control) and subsequently used for assays
of glycogen of the liver and uterine tissues.
Ethanol was administered intraperitoneally at a
dose of 3.0 g/kg body weight, as a 25% (V/V)
solution in saline. Epinephrine was injected
subcutaneously at a dose of 1 mg/kg body weight.
This hormone was prepared just before use by
diluting in saline with few drops of HCl to facilitate
dissolution, and its pH then being adjusted to 7.0.
Glucagon was administered intravenously at doses
of either 1µg or 10µg / mouse. The control groups
were injected with 0.9% saline to equate with the
volume of administered ethanol, epinephrine or
glucagon given intraperitoneally, subcutaneously or
intravenously, respectively. Animals were killed by
cervical dislocation either 2 min or 1 h after
treatment when the liver and uterus were
immediately excised and prepared for the assay of
glycogen.
Glycogen determination
Aliquots of tissue (0.5g) were placed in 2.5ml of
ice-cold 0.6 M perchloric acid, minced with fine
scissors, and homogenized using a polytron
followed by 2 or 3 strokes in a Dounce
homogenizer, glycogen was then assayed by the
method described by Bergmeyer (1984).
Immediately after homogenization, 2 lots of 0.2
ml of suspension were pipetted into separate
centrifuge tubes. One of these tubes served as a
glycogen sample, while the other served as a
glycogen sample, while the other served as a tissue
glucose blank. The tissue glucose blank was
centrifuged for 3 min in eppendorf centrifuge tubes
and 20 µl of the supernatant was retained for the
assay of glucose. With respect to the glycogen
sample, 20µl of homogenate was put in another
eppendorf tube with 100µl of KHCO3 and 200µl of
glucoamylase to hydrolyze the glycogen to glucose.
The sample was incubated at 40oC for 2 h, after
which time, 100ml of perchloric acid were added to
terminate the reaction. The samples were
centrifuged again for 3 min. in eppendorf tubes and
the supernatant retained for the assay of glucose.
Glucose determinations were conducted using
Boehringer glucose oxidase kits. The principle of
the test is:
Glucose + O2 + H20 Æ glukonate + H2O2
H2O2 + ABTS Æ coloured-complex + H2O.
After the addition of 5ml of glucose oxidase
reagent, the tubes was stood for 30 min. at room
temperature and absorbance were then read at
578nm.
RESULTS
The results presented in Figure 1 show that the
concentration of glycogen in endometrium changed
significantly with advancing pregnancy. The lowest
concentration was on day 3 (2.97 + 0.46), one day
before blastocyst implantation. The concentration
of the polysaccharide then increased through days 5
to 9 and then decreased slightly through to day 13.
Thus, the glycogen concentration in endometrium
increased almost 5 fold through days 3 to 9 of
pregnancy.
The pattern of glycogen concentration in the
whole uterus including both myometrium and
endometrium was similar to that of the
endometrium except that the levels of the
polysaccharide per gram of tissue were less in the
whole uterus than in the endometrium. However, in
the whole uterus, the glycogen concentration of the
tissue remained essentially unchanged through day
9 to 13 of pregnancy. These results indicate that
during this periods of pregnancy, glycogen is more
concentrated in the endometrium than in
myometrium.
 BioSMART Vol. 2, No. 1, hal. 1-6 3

15

Glycogen levels (umol/g or umol/uterus)

10

0
2 4 6 8 10 12 14
Days of pregnancy

Figure 1. Glycogen levels in the endometrium and whole uterus of the mouse during peri-and post-implantation pregnancy.
(Values represent the mean + S.E.M for N=5).

The results in Fig. 1 also show that the total pregnancy (Fig.2) add suggest that the
content of glycogen in the uterus was low through polysaccharide is produced continuously in the
days 3 to 7 of pregnancy, but increase progressively uterus over this period even after the time of
through days 9 to 13. However, over the period of initiation of decidual regression.
pregnancy studied, the uterine content of glycogen Figure 3 shows that the administration of
underwent more than 50-fold increase from an ethanol (3.0 g/kg body weight) to day 9-pregnant
initial value about 0.1143 µmol of glycosyl mice significantly decreased (P<0.001) glycogen
units/uterus on day 3 to approximately 6.0321 µmol concentration in the liver 1h after treatment.
of glycosyl units/g uterus on day 13. These changes Uterine glycogen occurred at lower concentration
in glycogen content relate to progressive increases than in liver and remained unaffected by exposure
in uterine weight during peri- and post-implantation to the acute dose of ethanol.

800
648.6

600
Uterine weight

385.4

400
258.2
140.8

200
104
60.4

0
3 5 7 9 11 13
Days of pregnancy

Figure 2. Changes in the weight of the uterus during peri- and post-implantation pregnancy in the mouse.
4 SUTARNO – Glycogenolysis 1. Hormonal Control

Ethanol

200

Glycogen concentration (umol/g)

116.18
Liver (Control)
***
100 Liver (Ethanol)

65.74
Uterus (Control)
Uterus (Ethanol)

10.73
9.97
0
Liver 1 Uterus

*** P<0.001, significantly different from control

Figure 3. Glycogen concentration (mmol of glycosyl units/g tissue) in liver and uterus of day 9-pregnant mice 1h after ethanol
administration (3.0g/kg body weight). Values represent the mean + S.E.M. for N=7.

The results presented in Fig. 4 show that The intravenous injection of glucagon at doses
subcutaneous administration of epinephrine of either 1 µg or 10 µg / mouse on day 9 of
(1mg/kg body weight) to day-9pregnant mice pregnancy significantly decreased (P<0.001) liver
decreased glycogen concentration in the liver glycogen concentration when measured 1h after the
significantly (P<0.001) 1h following the treatment. treatment, as shown in Figure 5. However, unlike
However, like ethanol, this hormone treatment the previous treatment, glucagon at a doses
failed to significantly alter the levels of glycogen in 10µg/mouse slightly decreased the uterine
the uterus, which occurred at much lower concentration of glycogen (P<0.05).
concentration than in the liver.
Epinephrine

200
Glycogen concentration (umol/g)

120.25

Liver (control)
100 Liver (Epinephrine)
*** Uterus (Control)
Uterus (Epinephrine)
49.46

10.58

10.73

0
Liver 1 Uterus

*** P<0.001, significantly different from control

Figure 4. Glycogen concentration (mmol of glycosyl units/g tissue) in the liver and uterus of day 9-pregnant mice 1h after
treatment with epinephrine (1mg/kg body weight). Values represent the mean + S.E.M. for N=7.
 BioSMART Vol. 2, No. 1, hal. 1-6 5

Glucagon 1 and 10 ug

200

Glycogen concentration (umol/g)

128.65
132.1
***
Liver (Control)

87.4
***
100 Liver (Glucagon)

64.58
Uterus (Control)
Uterus (Glucagon)

11.21
*
10.2

7.3

6.3
0
1ug glucagon1 administered 10 ug glucagon
2 administered
* = P<0.05; *** = P<0.001, significantly different from control

Figure 5. Glycogen concentration (mmol of glycosyl units/g tissue) in the liver and uterus of day 9-pregnant mice 1h after
treatment with glucagon at dose rates of either 1ug or 10 ug / animal. Values represent the mean + S.E.M. for N=7.
.

DISCUSSION large extent with the established growth pattern of

decidual cells (Finn and Porter, 1975), it is possible
It is evident from the results of the present study to suggest that these cells in the endometrium are
that glycogen levels in the mouse uterus increase the major sites of production of the polysaccharide.
significantly during peri- and post-implantation In this context, involution of the decidua in mice
pregnancy such that by day 13 the uterine content starts to occur after day 9 of pregnancy (Murdoch et
of the polysaccharide is more than 50-fold greater al., 1978), a time when the glycogen concentration
than on day 3. This production of glycogen of the uterus reached a maximum in the present
presumably provides optimal conditions for the study. However, the total content of glycogen
continued growth and development of the continued to increase after this time suggesting that
conceptus, since uterine glycogen has long been glycogen is still deposited in the uterus, but by cells
implicated as an important nutritional reserve for other than decidual cells which contribute to the
blastocyst implantation and embryonic continued growth of the uterus with advancing
development (Demers, et al., 1972), and parturition pregnancy. It is therefore apparent from this study
(Chew and Rinard, 1979). The energy requirements that glycogen is accumulated in endometrial
of the metabolic events surrounding nidation as decidual cells during early pregnancy at least until
well as uterine smooth muscle contractile processes the time of involution of the tissue. This may
are also believed to be satisfied in large part by a enable these cells to play a nutritive role during
rapid metabolization of glucose from uterine pregnancy, since glycogen, in tissues like liver and
glycogen reserve (Demers et al., 1972). skeletal muscle, is readily available energy store
The alteration in glycogen content and uterine which can be degraded on demand.
weight during peri- and post-implantation observed The mechanism whereby glycogenolysis is
in the present study are similar to those reported regulated in the uterus to provide glucose for the
previously in the rat uterus by Vasilenko et al., developing embryo remains uncertain. Ethanol has
(1981), in which the content of glycogen in the been reported to rapidly promote the degradation of
uterus (Myometrium + endometrium) increased glycogen to glucose in the liver (Winston and Reitz,
through days 5 to 15, remained high until the end of 1980; Simm and Murdoch, 1990), but not in the
pregnancy, and then decreased after parturition. uterus of the mouse during post implantation
Since the pattern of glycogen deposition in the pregnancy (Murdoch and Simm, 1992). The results
mouse uterus during pregnancy was consistent to a of the present study confirm these findings and
6 SUTARNO – Glycogenolysis 1. Hormonal Control
show that 1h after treating day 9-pregnant mice
with ethanol resulted in an increase of
glycogenolysis in the liver (almost 50% degraded),
but not in the uterus. This suggests that under
conditions of stress, uterine glycogen is conserved
to meet physiological demands other than those
required by the maternal system to cope with the
factors involved in this response.
In the present study, the administration of
glucagon to day 9-pregnant mice caused a decrease
in liver glycogen (almost 60%) 1 h after treatment,
and a small but significant decrease in uterine
glycogen. This slight effect on uterine glycogen
does not appear to be great enough to account for
the liberation of amounts of glucose that would
provide energy substrate to the uterus and / or
embryo sufficient for their support, and was only
apparent w3ith high levels of the hormone
(10µg/animal). Thus, the hormonal mechanism
facilitating regulation of glycogenolysis in the
uterus appears to be unique and is certainly
different from that operating in the liver.
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