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Hormonal control of endometrial glycogen metabolism in the Macaca arctoides.

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Hormonal Control of Endometrial Glycogen Metabolism
in the Marcarca arctoides
~
LAURENCE M. DEMERS, GORDON J. MACDONALD AND
ROY 0. GREEP
Laboratory of H u m a n Reproduction and Reproductive Biology, Department
of Anatomy, and T h e N e w England Regional Primate Research Center,
Harvard Medical School, Boston, Massachusetts 021 15
Endometrial biopsies obtained throughout the menstrual cycle
of the Macaca arctoides show the glycogen content paralleling the serum progesterone fluctuations which occur during the menstrual cycle. Secretory phase
samples contained a three-fold higher concentration of glycogen when compared
to follicular phase tissue. Changes in the activity levels of the glycogen metabolizing enzymes, glycogen phosphorylase and glycogen synthetase, during various stages of the menstrual cycle are in accord with the concept that the postovulatory increase in endometrial metabolism is a function of progesterone
influence on this tissue.
Endometrial glycogen synthetase activity remains low during the early proliferative phase of the cycle and becomes significantly elevated (two- to threefold) during the early secretory phase of the cycle. Glycogen phosphorylase
shows a similar cyclicity later in the luteal phase, reaching maximal activity between the seventeenth to nineteenth day of the cycle and remaining elevated
through the twenty-sixth day of the cycle.
The coincident nature of the rise in peripheral progesterone to increases in
uterine glycogen metabolism suggest that progesterone may be the prime modulator of uterine endometrial metabolism during the post-ovulatory phase.
ABSTRACT
Studies on the hormonal regulation of
endometrial metabolism in the subhuman
primate have been limited due to the difficulty in obtaining endometrial tissue
from the monkey on a serial basis. We
have recently described the rather unique
cervix uteri of the Macaca arctoides or
the Indochina bear monkey which has a
relatively straight endocervical canal.
Thus, serial endometrial biopsy sampling
is possible through routine curettage via
the cervical 0s (Demers et al., '72b). This
macaque demonstrates regular menstrual
cycles, a low incidence of seasonal
amenorrhea and its reproductive traits
compare favorably with other species of
macaques (Macdonald, '71). The studies
we wish to report describe the cyclicity of
endometrial glycogen metabolism in this
species as assessed by endometrial biopsy
throughout the course of the primate menstrual cycle with evidence to suggest a role
of progesterone in controlling endometrial
glycogenesis.
AM. J. PHYS.ANTHROP.,38: 183-188.
MATERIALS AND METHODS
This study employed mature female
Macaca arctoides caged individually in
temperature and light-controlled quarters
in the Laboratory of Human Reproduction
and Reproductive Biology. These monkeys
received Wayne monkey diet and tap
water ad libitum. Daily vaginal smears
established the length of the menstrual
cycle and the onset of menses.
Serum progesterone levels were determined based on the method described by
Neil1 et al., ('67). Blood samples were obtained by femoral venipuncture daily or
on alternate days in the morning. The
serum obtained following clotting and
centrifugation was extracted with petroleum ether and isolated by thin-layer
chromatography. I n this assay, male
M . arctoides serum was used for the source
of the binding protein. Tritiated progesterone ( N E N ) was used in all samples to
determine recovery of individual samples.
Uterine biopsies were obtained with a
183
184
L. M. DEMERS, G . J. MACDONALD AND R. 0. GREEP
Randall endometrial biopsy suction curette
which was carefully introduced into the
uterus until palpable at the fundus through
the anterior abdominal wall. All animals in
this study were anesthetized with sodium
pentobarbital 28.6 mg/kg administered IV.
For all biopsies, the perineal area was initially washed with Phisohex rinsed thoroughly with aqueous zephiran and aspirated dry to insure a clean vaginal well. All
animals were administered 300,000 units
of Bicillin (benzathine penicillin G suspension, Wyeth Labs., Phila., Penn.) following biopsy to safeguard against possible
infection.
Endometrial tissue samples were obtained throughout the menstrual cycle. In
all, 27 samples were obtained randomly
from six females over a 12-month period.
The tissue was frozen immediately in
liquid nitrogen and subsequently assayed
for gIycogen content, glycogen phosphorylase and glycogen synthetase activity.
Biochemical studies
Glycogen content. Tissue glycogen content was quantitated using the phenolsulfuric acid method a s described by Montgomery ('57). Results are expressed as
milligrams of glycogen/100 wet weight of
tissue.
Glycogen synthetase activity. Total synthetase activity was determined by following the incorporation of 14C glucose into
glycogen from uridine d i p h ~ s p h o - ~glu~C
cose in the presence of the cofactor glucose-6-phosphate (Demers et al., '72a). A
unit of enzyme activity was defined as 1
pmole glucose incorporated into glycogen
per gram wet tissue per hour.
Glycogen phosphorylase activity (total
and active form). Phosphorylase activity
was assayed according to Demers ('72a)
by following the incorporation of I4C glucose into glycogen from 14C glucose-l-phosphate in the presence ( t ) and absence ( a )
of the cofactor 5'AMP. A unit of enzyme
activity was expressed as /*mole glucose
incorporated into glycogen per gram wet
tissue per hour.
Total protein was determined by the
method of Lowry et al. ('51) with bovine
serum albumin as a standard.
RESULTS
That ovarian steroids control endometrial
metabolism in the human female has been
well documented. The metabolic changes
in endometrial tissue throughout the
course of the menstrual cycle are in response to the controlling influence of the
ovarian steroid secretion pattern. These
studies with endometrium from the subhuman primate, Macaca arctoides show
the direct relationship of glycogen metabolism to the peripheral blood titer of progesterone. Figure 1 depicts the composite
relationship between the serum progesterone values and the endometrial glycogen
levels throughout the course of the menstrual cycle. Both these parameters exhibit a significant increase at approximately day 12 of the cycle and reach a
pinnacle about day 18 followed by a decline on about day 23-25 which continues
until menses.
The activity pattern of the enzyme system glycogen synthetase believed to be rate
limiting to glycogen synthesis was determined in this endometrial tissue and
parallels closely the deposition pattern of
glycogen at various stages of the cycle
(fig. 2 ) . A significant increase occurs
about day 12, peaks about days 17 to 19
and then declines during the late secretory
phase of the cycle.
Both the total and active forms of the
glycogen phosphorylase enzyme, the enzyme systems which control the breakdown of this glucose polymer, have activity patterns similar to both glycogen
deposition and the increase in glycogen
synthetase activity. These increase about
day 12 of the cycle (fig. 3 ) . The phosphorylase activity pattern, however, differs
somewhat from the synthetase enzyme in
that during the late secretory phase of the
cycle, it remains elevated through day 27
of the cycle. This was the case regardless
of the enzyme form, active or total. The
activity curves for both enzymes were calculated for specific activity during each of
these phases of the monkey cycle and the
activity pattern was similar to that expressed per gram wet weight of tissue.
DISCUSSION
Our understanding of the physiology of
menstruation in the subhuman primate as
185
PRIMATE ENDOMETRIAL GLYCOGEN METABOLISM
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DAYS
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12 14 16 18 20
OF MENSTRUAL CYCLE
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Fig. 1 Mean serum progesterone levels ( k S E ) from seven Macaca arctoides and mean
endometrial tissue glycogen values ( k SE) from 59 biopsy samples assessed during various
phases of the menstrual cycle. Progesterone values are expressed in nanograms per ml
serum while the glycogen values are expressed in milligrams of glycogen per 100 gm of wet
weight of tissue.
well as the cyclicity of endometrial
changes during the primate menstrual
cycle are largely due to the pioneer efforts
of Allen ('27), Bartelmez ('51), Corner
('35) and Hisaw ('35). These investigators advanced our knowledge of endometrial growth, proliferation and secretory
activity on a morphologic basis with evidence to suggest ovarian steroid control of
these changes. Little information is available, however, a s to how the steroid hormones, estrogen and progesterone, effect
their control at the molecular level. Our
results demonstrate that in the macaque
as in the human female, there is a cyclicity
of endometrial glycogen deposition. This
shows a significant increase about day 12
of the menstrual cycle, has a n apex in
concentration about day 17 then declines
continuously to menses. The coincident
rise in progesterone is strikingly similar to
the glycogen concentration pattern and
suggests that progesterone may control endometrial glycogenesis. That progesterone
is capable of promoting a marked increase
in the glycogen content of human endometrial explants cultured in vitro in progesterone supplemented medium has been demonstrated previously (Hughes et al., '69).
The activity patterns of the glycogen metabolizing enzymes, glycogen synthetase
and glycogen phosphorylase in primate en-
186
L. M. DEMERS, G . J. MACDONALD AND R. 0. GREEP
onv of crctt
Fig. 2 The activity of total glycogen synthetase in endometrium from M. arctoides during
various phases of the menstrual cycle. Values are expressed as micromoles glucose incorporated into glycogen per gram of wet tissue per hour. Each point represents mean
value & SE.
DAYS OF MENSTRUAL CYCLE
Fig. 3 The activity of endometrial glycogen phosphorylase in the M. arctoides during
various phases of the menstrual cycle. Enzyme activity determined in the absence ( - AMP, a
Form) and presence ( + AMP, to Form) of the cofactor 5' adenosine monophosphate. Values
represented as mean 2 SE at each point are expressed as micromoles glucose incorporated
into glycogen per gram of wet tissue per hour.
PRIMATE ENDOMETRIAL GLYCOGEN METABOLISM
dometrium parallel the changes in tissue
glycogen and are most probably responsible for the changes observed. Whether
progesterone causes induction and/or
activation of these specific enzyme processes is uncertain at this time. Further
studies are underway in our laboratory. It
is suggestive, however, by virtue of the
coincident rise in serum progesterone and
endometrial glycogen, that progesterone is
involved in stimulating endometrial glycogenesis in estrogen-primed tissue in the
subhuman primate.
ACKNOWLEDGMENTS
The authors wish to express their gratitude to Miss Jean Greenbaum and Miss
Johanna McCann for their technical skills
and to Mrs. Pauline Breen for typing the
manuscript,
Supported by NIH HD 03736, The Ford
Foundation and The Lalor Foundation.
LITERATURE CITED
Allen, E. 1927 The menstrual cycle of the
monkey, Macacus Rhesus: Observations on
normal animals, the effects of the removal of
the ovaries and the effects of injections of
ovarian and placental extracts into the spayed
animals. Contr. Embryol. Carnegie Inst., Washington, 19: 1 4 4 .
Bartelmez, G. W. 1951 Cyclic changes in the
endometrium of the rhesus monkey (Macaca
187
mulatta). Contr. Embryol. Carnegie Inst.,
Washington, 34: 101-144.
Corner, G. W. 1935 Influence of the ovarian
hormones, oestrin and progestin, upon the
menstrual cycle of the monkey. Am. J. Physiol.,
11 3: 238-250.
Demers, L. M., S. G. Gabbe, C. A. Villee and
R. 0. Greep 1972a The effect of insulin on
human placental glycogenesis. Endocrinology,
91: 270-275.
Demers, L. M., G. J. Macdonald, A. T. Hertig,
N. W. King and J. J. MacKey 1972b The
cervix uteri in Macaca mulatta, Macaca
arctoides and Mncaca fascicularis - a comparative anatomic study with special reference
to Mncaca arctoides as a unique model for endometrial study. Fertil. Steril., 23: 519-534.
Hisaw, F. L. 1935 The physiology of menstruation in Macacus rhesus monkeys. Am. J. Obst.
& Gynec., 29: 638-659.
Hughes, E. C., L. M. Demers, T. Csermely and
D. B. Jones 1969 Organ culture of human
endometrium. Effect of ovarian steroids. Amer.
J. Obst. & Gynec., 105: 707-720.
Lowry, 0. H., W. J. Rosebrough, A. L. Farr and
R. ,T. Randall 1951 Protein measurement
with- the Folin phenol reagent. J. Biol. Chem.,
193: 265-275.
Macdonald, G. J . 1971 Reproductive patterns
of three species of macaques. Fertil. Steril., 22:
373-377.
Montgomery, R. 1957 Determination of glycogen. Arch. Biochem. Biophys., 67: 378-386.
Neill, J. D., E. D. B. Johansson, J. K. Datta and
E. Knobil 1967 Relationship between the
plasma levels of luteinizing hormone and progesterone during the normal menstrual cycle.
J . Clin. Endocr., 27: 1167-1173.
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endometrial, metabolico, hormonal, macaca, glycogen, control, arctoides
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