Uterine Size and Glycogen Content in Cycling and Pregnant. Rats: Influence of Relaxin

Transcription

1 BIOLOGY OF REPRODUCTION 25, (1981) Uterine Size and Glycogen Content in Cycling and Pregnant. Rats: Influence of Relaxin PETER VASILENKO Ill, WALTER C. ADAMS2 and EDWARD U. FRIEDEN Department of Biological Sciences and Department of Chemistry, Kent State University, Kent, Ohio ABSTRACT Uterine weight and glycogen content were measured at stages of the estrous cycle and in the gravid and nongravid horns of unilaterally pregnant rats. The response of these parameters to administration of a purified preparation of porcine relaxin (relaxin B) was assessed during the estrus-diestrus interval and at Day 15 of gestation. Both uterine weight and glycogen content were greatest at proestrus and estrus and lowest at diestrus-1; when 0.1 mg of relaxin was administered beginning at estrus and injected over a 24 h period, the decline in uterine weight and glycogen was retarded. During gestation uterine weight declined prior to implantation and increased rapidly thereafter to a peak at Day 22 beyond which uterine size decreased through parturition. Uterine glycogen content followed similar increases except that the peak occurred on Day 23 and was sharply defined in advance of parturition at which time a rapid decline was observed. The increases in uterine weight and glycogen were significantly greater in the gravid horn than in the nongravid horn, and on the day of parturition both horns exhibited the highest glycogen concentration in the myometrium alone. Injection of relaxin at Day 15 of gestation increased uterine weight and glycogen content significantly in the myometrium of the nongravid horn without affecting the highly variable parameters in the gravid horn. Since uterine glycogen and tissue growth profiles parallel recently reported RIA relaxin concentrations in rats rather than changes in steroid hormone levels, these data suggest that relaxin may play a significant role in the uterine accommodation and glycogen storage which occur during late gestation. INTRODUCTION The uterus undergoes marked changes in size and composition during normal reproductive cycles and during gestation. In the rat, uterine glycogen, an important energy source for both embryonic development (Demers et al., 1972) and parturition (Chew and Rinard, 1979), reaches its highest concentration at proestrus, decreases during estrus, and is lowest at diestrus (Boettiger, 1946; Walaas, 1952). Likewise, during early pregnancy, uterine glycogen concentration gradually decreases from the initial estrus values (Demers et al., 1972) and then increases again some 2-6 fold during late pregnancy (Kostyo, 1957; Parvez and Parvez, 1974; Accepted April 6, Received January 13, Present address: Case Western Reserve University, School of Medicine, Dept. of Reproductive Biology, MacDonald House-7, University Hospitals, 2105 Addbert Road, Cleveland, OH Reprint requests. Chew and Rinard, 1979). Uterine tissue is also enlarged during proestrus and estrus, although partially due to accumulation of water (Boettiger, 1946; Walaas, 1952), and during gestation the uterus expands many times beyond its nongravid size to accommodate the growing conceptus, returning to nearly its former size and condition following parturition (Reynolds, 1965). Taken together, the increased glycogen concentration and uterine weight during gestation mean that the uterus produces and stores great quantities of carbohydrate during a relatively short period. Alterations in uterine size and metabolism correspond to variations in estrogen and progesterone concentrations (Yoshinaga et al., 1969; Pepe and Rothchild, 1974). However, the secretory patterns of these steroids may not account fully for the magnitude of the tissue changes which occur during gestation. We have shown (Vasilenko et al., 1980) that purified relaxin preparations can produce increases in uterine glycogen and tissue mass in ovariectomized, immature rats in a dose-dependent 162

2 UTERINE SIZE AND GLYCOGEN CONTENT IN THE RAT 163 fashion with or without estrogen priming although estrogen augments the response. It appears that both the uterotropic and glycogenic properties of relaxin, at least in the rat, are inherent to the hormone. The concentrations of relaxin in plasma of pregnant rats have been measured by a radioimmunoassay specific for rat relaxin (Sherwood et al., 1980) and show a gradual rise during the latter half of gestation and a surge prior to parturition. In an attempt to correlate changes in glycogen concentrations and uterine mass with these data, we have made a systematic study of these parameters during gestation, compared them with fluctuations seen during the estrous cycle, and tested the responsiveness of the tissue to purified porcine relaxin during gestation. Unilaterally pregnant rats were utilized to examine the changes in the gravid uterus under local influence of fetal units and the systemic effects on the nongravid uterus in their absence. Our data support the hypothesis that relaxin may be a complement of the ovarian steroids in the control of uterine growth and metabolism during gestation. MATERIALS AND METHODS Rats used in these experiments were virgin Sprague-Dawley females bred and housed in a controlled environment with a 12L:12D lighting schedule (lights on h). At 70 days of age daily vaginal smears were begun, and following the establishment of three to four consecutive 4 day cycles rats were either mated at proestrus or killed at specific estrous stages. Rats were prepared for unilateral pregnancy by ligating the right oviduct under ether anesthesia \ 2 weeks prior to scheduled mating. Day I of pregnancy was designated as the day in which sperm were observed in the vaginal smear; parturition most commonly occurred on Day 23 of gestation. Pregnant rats on Days 5, 10, 15, 20, 22, 23, and postpartum Day 1 and cycling rats in proestrus, estrus, and diestrus (Day 1) were killed by cervical dislocation and exsanguinated. All animals were fasted for 24 h prior to autopsy and were killed between 0800 and 0900 h. In cycling animals the uteri were removed, weighed, and each horn divided in half longitudinally; one-half of each uterus was analyzed for glycogen content with the endometrium present and the other half after the endometrium was quickly scraped away Purification of porcine relaxin yields three dcctrophorectically distinct peptides with relaxin activity. The peptide used in this experiment was relaxin B which appears to be identical with relaxin CM-B (Sherwood and O Byrne, 1974) and the preparation sequenced by Schwabe and McDonald (1977). with a sharp blade at room temperature and discarded. In pregnant animals the gravid and nongravid uteri were removed and separated at the cervical region; the gravid horn was split longitudinally, emptied of conceptuses, and the decidua excised for separate glycogen determination. Both gravid and nongravid organs were then divided in half and analyzed with or without endometrium present. Within 5 mm of death glycogen extraction was begun, and content was determined after the method of Walaas (1952) as previously described (Vasilenko et al., 1980). The effects of a purified porcine relaxin (Fraction B )3 with a potency of 1800 GPU/mg was measured in diestrus (Day 1) and Day 15 pregnant rats. Beginning at 0800 h, 24 h after proestrus or Day 14 of gestation, animals were injected s.c. every 6 h with 25 g of relaxin in 0.2 ml saline for a total dose over 24 h of 0.1 mg, and were killed between 0800 and 0900 h the following morning. Diestrus was confirmed by vaginal smear at the time of autopsy. Means of all data were compared by Student s t test, and statistical significance reported with a probability level of 0.01 or less unless otherwise noted. RESULTS Uterine weights during proestrus and estrus were similar and declined some 30% (P<0.01) to the diestrus-1 value (Fig. 1). The endometrium contributed insignificantly to these variations. Relaxin B at a total dose of 0.1 mg increased uterine weight significantly (P<0.01), nearly to the level of the estrus uteri. Uterine glycogen concentrations were also the same during proestrus and estrus but decreased by more than one-half in diestrus (P<0.01). Although relaxin significantly increased uterine glycogen concentration from the expected diestrus level or retarded its decline from estrus, the tissue content was maintained at only values intermediate between diestrus and estrus uteri. Calculation of total glycogen content revealed a decrease of more than two-thirds (P<0.01) from estrus to diestrus but of only 40% when relaxin was administered, a significant change (P<0.01) from nontreated uteri. After 5 days of gestation, the weight of the gravid uterus decreased somewhat (24%; P<0.01) and then progressively increased some 10-fold during Days 10 to 12 (Fig. 2). A slight decrease occurred from Day 22 to the day of parturition; subsequently the uterine weight decreased sharply by more than one-third during the next 24 h. Changes in weight of the endometrium and myometrium alone display a pattern similar to that of the total uterus, and the relative contribution of each layer to the total uterine weight remained constant throughout gestation. The weight of the decidua

3 164 VASILENKO III ET AL. o ENDOMETRIUM + MYOMETRIUM FIG. I. Uterine weight, glycogen concentration, and total tissue glycogen in rats at three stages of the estrous cycle and following injection of 0.1 mg of relaxin B given in four doses of 25 pg each every 6 h. P = proestrus; E = estrus; D1 = diestrus Day 1; D + R = diestrus-i animals which had been exposed to relaxin the previous 24 h. Each group contained seven animals and the vertical bars indicate SEM. reached a peak at Day 20, remained essentially unchanged for the next 24 h, and then decreased rapidly during the next 2 days; overall, the development of the decidua was somewhat out of phase with the growth of the uterus as a whole. Litter size did not vary among groups and averaged seven pups. The nongravid horn followed a pattern of weight change similar to that of the gravid uterus, both for the total tissue and myometrium alone. The maximal increase in weight, while significant (P<0.01) was only 59% above estrus values. Uterine glycogen concentration in the gravid uterus decreased from Day 1 to Day 10 of gestation (P<0.01) and then increased steadily over the next 12 days (Fig. 3). Its concentration then tripled from Day 22 to Day 23, the 24 h immediately preceeding parturition, and dropped again to levels intermediate between diestrus and estrus during the succeeding 24 h (postpartum). The glycogen content of the nongravid uterus followed the same trend of gradual increase to Day 22 followed by a sharp spike on Day 23 and a return to resting levels although the magnitude of the change was only one-half that in the gravid uterus. In both the gravid and nongravid horns the presence of the endometrium had no significant effect on glycogen concentration until the day of birth, when the myometrium alone exhibited a significantly higher glycogen level (P<0.01). The decidua, on the other hand, showed an entirely different profile, similar to that of weight changes in this tissue, with glycogen concentrations on Day 15 some 3 times the level in the myometrium and a comparatively slight but significant further rise to Day 20 followed by a decline prior to parturition so that at that time myometrial glycogen was twice the concentration in the decidua. Relaxin, administered to unilaterally pregnant rats from Day 14 to 15, produced significant increases (P<0.05) in myometrial weight (26%) and myometrial glycogen concentration (3 5%) of the nongravid uterus (Fig. 4). Thus the total glycogen content in the myometrium rose 67% (P<0.01). The presence of the endometrium eliminated the significant increase in glycogen concentration in the nongravid horn, while in the gravid horn weight and glycogen values were extremely variable and no significant effects were noted. DISCUSSION Uterine weight and glycogen concentration are lowest during diestrus and highest during proestrus and estrus as reported previously (Boettiger, 1946; Walaas, 1952). As gestation proceeds both uterine size and glycogen content decline from the initial estrus levels, suggesting an increased glycogen utilization during the critical period of implantation and decidualization (Demers et al., 1972). Kostyo (1957) reported that in the pregnant rat glycogen in the uterus does not exceed that found at proestrus whereas Chew and Rinard (1979) found

4 UTERINE SIZE AND GLYCOGEN CONTENT IN THE RAT 165 GRAVID HORN OTotal Horn Q-QMyometriUm + Endometrium L-Myometrium D-D Decidua b 2000 E I- I CD 1000 z w NOW-(2RAVIfl HORN P -i DAY OF GESTATION FIG. 2. Uterine weights of the gravid and nongravid horns of unilaterally pregnant rats. P = parturition; -1 = Day 1 postpartum. Each point represents the mean of six rats ± SEM. When a symbol is larger than the SEM, the SEM is not given. that glycogen concentration rose to more than twice that of the proestrus uterus immediately before parturition. However, the earlier investigation reported glycogen levels measured in the nongravid horn of unilaterally pregnant rats while the latter one compared glycogen content of the myometrium of pregnant uteri. Our data show that glycogen concentration increases throughout the second half of pregnancy in both the gravid and nongravid horns and increases sharply on the day of parturition to levels well above those found in proestrus or estrus uteri. The 10-fold greater response seen in the gravid as compared with the nongravid horn suggests that either the presence of the conceptus or endocrine effects generated by it or the ipsilateral ovary may be of importance and may explain discrepancies between the earlier reports. Glycogen deposition in the uterus appears to be limited to the myometrium in estrogentreated rats (Bo and Atkinson, 1952; Walaas, 1952) and mice (Hall, 1960, 1965, 1966). We have found that the presence of the endometrium does not affect glycogen measurements throughout gestation except on the day of parturition, when significantly greater glycogen levels were observed in the myometrium alone. The rise and fall of glycogen levels at term are consistent with a role of glycogen as an energy resource for the parturient uterus (Chew and Rinard, 1979). During gestation the uterus undergoes extensive modification to accommodate and support

5 166 VASILENKO III ET AL GRAVID HORN O-O Myometrium + Endometrium L3-5 Myometrium C E 1OOO DECIDUA 180C 60O 4cx C.) Z20 0 C., NON-GRAVID HORN Total Horn A-A Myometrlum DAY OF GESTATION FIG. 3. Uterine glycogen concentrations in the gravid and nongravid horns of unilaterally pregnant rats. P = parturition; -1 Day 1 postpartum. Each point represents the mean of six rats ± SEM. When a symbol is larger than the SEM, the SEM is not given. the developing fetus. Initially, a slight decline in weight occurs prior to implantation after which the uterus gradually enlarges to permit growth of the fetus. During the second half of pregnancy the uterus grows rapidly as fetal growth accelerates; however, in the 3 days preceding parturition the rate of uterine growth declines, and fetal growth is accommodated by stretching of the uterus (Reynolds, 1965). Both the nongravid and gravid uterus follow much the same pattern but with a different magnitude, suggesting that both systemic and local influences are operating. Following parturition both uteri shrink rapidly to a fraction of their peak sizes. Uterine growth and glycogen accumulation during gestation occur in the presence of estrogen, progesterone, and relaxin. During pregnancy, plasma estrogen levels are generally low (much more so than during the estrous cycle) except near term when a rapid increase is observed, and levels comparable to those found during proestrus are reached on the day of parturition (Yoshinaga et al., 1969). Estrogen is known to increase uterine weight and glycogen deposition (Boettiger, 1946; Walaas, 1952; Kostyo, 1957; Bo and Atkinson, 1952;Demers et al., 1972; Demers and Jacobs, 1973; Bo et al., 1973). However, the low levels of estrogen between Days 10 and 22 of pregnancy are insufficient to account for the increases in uterine weight and glycogen concentration

6 UTERINE SIZE AND GLYCOGEN CONTENT IN THE RAT 167 NON-GRAVID HORN: MYOMETRIUM E ENDOMETRIUM + MYOMETRIUM U;: DECIDUA GRAVID HORN: I- I C, Lu Ui z iu I- 15d 15d+R 15d 15d+R 15d 15d+R FIG. 4. Effect of relaxin (0.1 mg total dose) on uterine weight, glycogen concentration, and total glycogen in the gravid and nongravid horns of unilaterally pregnant rats at Day 15 of gestation. 15d = Day 15 rats; 15d + R = rats receiving relaxin in four doses (25 pg in 0.2 ml saline) at 24, 18, 12, and 6 h prior to sacrifice. Each group comprises six rats, and vertical lines indicate SEM. which occur at this time. Also, while plasma estrogen at parturition may reach only proestrus levels, uterine glycogen concentration is some 4.5 times the proestrus value. Progesterone concentrations increase gradually during the first half of pregnancy, but peak and thereafter dedine at 15 days in the rat, falling to low levels in the 3 days prior to parturition (Pepe and Rothchild, 1974). This hormonal profile, plus the lack of significant regulatory effect of progesterone on uterine glycogen (Walaas, 1952; Bo and Atkinson, 1952; Demers et al., 1972, Cecil and Bitman, 1968; Garrison et al., 1973) cannot account for the preparturient weight and glycogen gain. However, progesterone concentration in the blood does correlate closely with the development and glycogen content of the dccidua (Pepe and Rothchild, 1974) and may control this aspect of the pregnant uterus. Decidua,

7 168 VASILENKO Ill ET AL. containing large quantities of glycogen, may act as an energy reserve for the developing embryos. The uterine glycogen profile closely parallels the plasma relaxin levels reported in pregnant rats based on radioimmunoassay (Sherwood et al., 1980). From Day 1 to 10, when uterine glycogen declines, no relaxin is detectable in ovaries or serum. Relaxin levels increase between Days 10 and 20 and a surge is seen at term, followed by a postpartum decline. The period of myometrial growth required for accommodation of fetal development thus coincides with plasma relaxin levels much more closely than with estrogen titers. Relaxin has been shown to increase uterine weight and glycogen in ovariectomized immature rats (Steinetz et al., 1957; Kroc et al., 1959; Vasilenko et al., 1980), and glycogen increases specific to the myometrium of intact and ovariectomized mice have been observed histochemically following relaxin treatment (Hall, 1960, 1965). In the present study, relaxin treatment significantly increased uterine weight and glycogen content in intact, cycling rats and in the nongravid uteri of unilaterally pregnant rats. The lack of a significant response to exogenous relaxin in the gravid uterus suggests the dominance of the local hormonal environment in regulating growth and metabolism in this tissue, at least at 15 days of gestation. We have also shown (Frieden et al., 1980) that protein content and incorporation of labeled leucine is enhanced by relaxin (although this has not been tested in the uterus of the pregnant rat), findings associated with the growth response. We propose, therefore, that relaxin contributes importantly to the accumulation of uterine tissue and stored glycogen that occurs during late gestation, and synergizes with estrogen to account for changes that occur in the absence of amounts of the steroid adequate alone to produce uterine accommodation. The mechanism of this effect of relaxin could be either an augmentation of glycogen synthesis or a glycogen sparing effect which may be related to myometrial relaxation (Sawyer et al., 1953; Wiqvist and Paul, 1958; Griss et al., 1967). Experiments are currently underway to explore which of these mechanisms is the more likely and the degree to which relaxin can contribute to the hormone replacement necessary to maintain gestation in ovariectomized rats. REFERENCES Bo, W. J. and Atkinson, W. B. (1952). Histochemical studies on glycogen deposition in the uterus of the rat. I. In intact cyclic animals and in castrates treated with ovarian hormones. Anat. Rec. 113, Bo, W. J., Krueger, W. A. and Garrison, B. M. (1973). Effect of repeated injection of oestrogen on uterine glycogen. J. Endocrinol. 59, Boettiger, E. C. (1946). Changes in the glycogen and water content of the rat uterus. J. Cell. Comp. Physiol. 27, Cecil, H. C. and Bitman, J. (1968). Effect of steroid hormones on estrogen induced uterine glycogen synthesis. J. Endocrinol. 42, Chew, C. S. and Rinard, C. A. (1979). Glycogen levels in the rat myometrium at the end of pregnancy and immediately postpartum. Biol. Reprod. 20, Demers, L. M. and Jacobs, R. D. (1973). Hormonal regulation of rat uterine glycogen metabolism. Biol. Reprod. 9, Demers, L. M., Yoshinaga, K. and Greep, R. 0. (1972). Uterine glycogen metabolism of the rat in early pregnancy. Biol. Reprod. 2, Frieden, E. H., Vasilenko, P. and Adams, W. C. (1980). Effects of purified relaxin on glycogen and protein contents of the rat uterus. In: Relaxin. Proceedings of the 15th Midwest Conference on Endocrinology and Metabolism. Plenum Press, New York. In Press. Garrison, B. M., Bo, W. J. and Krueger, W. A. (1973). The influence of estradiol-17j3 dipropionateprogesterone ratios on uterine glycogen in the rat. Steroids 22, Griss, C., Keck, j., Engelhorn, R. and Tuppy, H. (1967). The isolation and purification of an ovarian polypeptide with uterine-relaxing activity. Biochim. Biophys. Acts 140, Hall, K. (1960). Modification by relaxin of the response of the reproductive tract of mice to oestradiol and progesterone. J. Endocrinol. 20, Hall, K. (1965). l-listochemical investigation of the effects of oestrogen, progesterone and relaxin on glycogen, amylophosphorylase, transglycosylase and uridine diphosphate glucose-glycogen glucosyl transferase in uteri of mice. J. Endocrinol. 32, Hall, K. (1966). A histochemical study of enzymes concerned in glycogen metabolism in the myometrium of pregnant and parturient mice. J. Endocrinol. 34, Kostyo. J. L. (1957). A study of the glycogen levels of the rat uterus and certain skeletal muscles during pregnancy. Endocrinology 60, Kroc, R. L., Steinetz, B. C. and Beach, V. L. (1959). The effects of estrogens, progestagens, and relaxin in pregnant and nonpregnant laboratory rodents. Ann. NY Acad. Sci. 75, Parvez, S. and Parvez, H. (1974). Glycogen storage during the last part of pregnancy in uterus, liver and heart of rats. Experientia 30(11), Pepe, G. J. and Rothchild, I. (1974). A comparative study of serum progesterone levels in pregnancy

8 UTERINE SIZE AND CLYCOGEN CONTENT IN THE RAT 169 and in various types of pseudopregnancy in the rat. Endocrinology 95, Reynolds, S.R.M. (1965). Patterns of uterine growth during pregnancy. In: Physiology of the Uterus. (S.R.M. Reynolds, ed). Hafner Publishing Co., New York. pp Sawyer, W. H., Frieden, E. H. and Martin, A. C. (1953). In vitro inhibition of spontaneous contraction of the rat uterus by relaxin-containing extracts of sow ovaries. Am. J. Physiol. 172, Schwabe, C. and McDonald, J. K. (1977). Relaxin: A disulfide homolog of insulin. Science 197, Sherwood, 0. D. and O Byrne, E. M. (1974). Purification and characterization of porcine relaxin. Arch. Biochem. 160, Sherwood, 0. D., Crnekovic, V. E., Gordon, W. L. and Rutherford, J. E. (1980). Radioimmunoassay of relaxin throughout pregnancy and during parturition in the rat. Endocrinology 107, Steinetz, B. C., Beach, V. L., Blye, R. P. and Kroc, R. L. (1957). Changes in the composition of the rat uterus following a single injection of relaxin. Endocrinology 61, Vasilenko, P., Frieden, E. H. and Adams, W. C. (1980). Effect of purified relaxin on uterine glycogen and protein in the rat. Proc. Soc. Exp. Biol. Med. 163, Walaas, 0. (1952). Effects of oestrogens on the glycogen content of the rat uterus. Acts Endocrinol. 10, Wiqvist, N. and Paul, K. (1958). Inhibition of the spontaneous uterine motility in vitro as a bioassay of relaxin. Acta Endocrinol. 29, Yoshinaga, K., Hawkins, R. A. and Stocker, J. F. (1969). Estrogen secretion by the rat ovary in vivo during the estrous cycle and pregnancy. Endocrinology 85,