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 400 M A C A C A ARCTOIDES "= 7 A - 2 ._ I 0 m 0 I00 5! $ I- z YI 200 t3 f0 00 $ > 0 ? - 4 6 DAYS 8 10 12 14 16 18 20 OF MENSTRUAL CYCLE 22 24 26 28 30 . 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.