Postpartum regeneration of the guinea pig endometriumRelationship to serum estradiol and progesterone concentrations.код для вставкиСкачать
THE ANATOMICAL RECORD 210:41-44 (1984) Postpartum Regeneration of the Guinea Pig Endometrium: Relationship to Serum Estradiol and Progesterone Concentrations DAVID R. GARRIS Department of Anatomy, East Carolina University School of Medicine, Grienuille, NC 27834 ABSTRACT The pattern of cell mitotic activity in the uterus of postpartum, pregnant guinea pigs was correlated with the associated changes in serum progesterone and estradiol levels between days 1-8 of pregnancy. Stromal and glandular epithelial mitotic patterns mimicked the associated fluctuations in serum progesterone and estradiol levels, respectively. No changes in myometrial mitotic activity were observed. The luminal epithelial mitotic index was apparently sensitive to the relative change in the ratio of progesterone-to-estradiol in circulation. These studies indicate that postpartum, endometrial preparation for blastocyst implantation depends on the proper priming of the uterus by both progesterone and estradiol in the guinea pig. In order for blastocyst implantation to SUCceed, the uterine endometrium must be properly prepared and sensitized to support subsequent differentiation and growth of the conceptus (Psychoyos, 1973; Finn, 1977) by ovarian steroid hormones. In virgin or nongravid females, estrogen serves to stimulate uterine epithelial and glandular growth, whereas progesterone apparently subserves these functions relative to stromal preparation for implantation (Finn, 1977). However, in the postpartum pregnant female, the endometrial trauma induced by parturition necessitates that both epithelial and stromal growth be reestablished, in additon to proper hormonal conditioning of the endometrium, in order for a subsequent implantation reaction to occur. Considering that in the rat and guinea pig blastocyst implantation occurs at approximately 6 days postovulation, the endometrial changes that occur in postpartum pregnant animals must be rapid and dramatic with respect to cellular reorganization. Since previous studies with the guinea pig have indicated that the estrogen-induced, uterine hyperemia associated with the nidatory process in this species is present in the postpartum pregnant female (Garris and Whitehead, 1981), it was of interest to determine the pattern of stromal, glandular, and epithelial cell growth relative to the cyclic changes in circulating progesterone and estradiol levels in this model system. The fol- 0 1984 ALAN R. LISS, INC. lowing study was undertaken to determine the pattern of cell mitotic activity in the postpartum, pregnant guinea pig uterus relative to the timing of blastocyst implantation and circulating ovarian steroid hormone levels. MATERIALS AND METHODS Adult female guinea pigs (Dunkin-Hartley) weighing between 600 and 1,000 g were used in this study. All animals were maintained under a controlled photoperiod of 14 hr light/ day (lights on 0600 hr) with food and water available ad libitum. Breeding was accomplished by placing a postpartum female with a breeder male overnight. The use of this model in our colony increases the rate of successful breedings to nearly loo%, with day 1 of pregnancy denoted by either the presence of sperm in the vaginal lavage or a sperm plug. Females were then removed to a separate cage until used. Between 1000 and 1200 h r on days 1-3 or 5-8 postpartum, each animal received a n IP injection of colchicine (4mgkg) at 2 hr prior to sacrifice. Blood samples were collected by either direct cardiac puncture or by decapitation a t the end of each experiment. Blood was allowed to clot and stored at 5°C overnight, and the serum was collected following centrifugation and stored a t -20°C until asReceived November 15, 1983;accepted March 26, 1984 42 D.R. GARRIS sayed. Serum 170-estradiol (E) and progesterone (P) levels were estimated by radioimmunoassay as previously described (Garris and Whitehead, 1981) using E antibody GDN244 and P antibody GDN337. All samples were run in duplicate and are expressed uncorrected for procedural loss ( < 5%). Intraassay and interassay variability was less than 5 and 8%, respectively. The specificity of the P antibody has been previously described (Gibori et al., 1977), with assay sensitivity ranging between 0 and 30 ngl ml. The specificity of the E antiserum has been reported (Korenman et al., 1974). Uterine horns were removed from each female guinea pig and prepared for histological analysis using conventional techniques for paraffin-embedded tissue samples. Mid-uterine segments that did not possess placental scars were serially sectioned (8 pm) and stained with hematoxylin and eosin. The number of epithelial, glandular, stromal, and myometrial cells that were observed to be arrested in the metaphase stage of mitosis were counted from 3-5 random sections of each uterus. All data were analyzed as group means (k SEMI. Intergroup differences were analyzed using the Student’s t-test with a significance level set a t P < 0.05. RESULTS The changes in the mitotic index of epithelial, glandular, stromal, and myometrial cells in the postpartum, pregnant guinea pig uterus are depicted in Figure 1. The epithelial cell mitotic activity was elevated between days 1-5 postpartum and subsequently declined between days 6-8. The mitotic pattern of glandular epithelial cells exhibited a decline in activity between days 1-3 postpartum followed by a peak in cellular division rate on day 5 postpartum. Subsequently, the mitotic index of the glandular cells declined through day 8 postpartum to basal rates. Stromal cells exhibited minimal mitotic activity between days 1-3 postpartum, after which time a rapid rise to peak division rates was observed between days 5-7. This rise in stromal mitotic activity was temporally associated with the timing of blastocyst implantation in this species (Garris, 1984a), and rapidly declined to near basal rates by day 8 postpartum. The myometrial mitotic index remained relatively constant between days 1-8 postpartum with no significant changes observed relative to the timing of blastocyst implantation in these animals. 301 801 60 l a n’ ma 5 6 7 8 D a y Post-Partum Fig. 1. The mean ( * SEM) number of mitotic cells in the luminal epithelium, glandular epithelium, stroma, and rnyometrium of the postpartum, pregnant guinea pig uterus is represented. The number of animals per group is indicated in parentheses. Intergroup differences are denoted as a vs. b, b vs. c, or c vs. d: P 4 0.05; a vs. c: P < 0.01; a va. d: P < 0.001. The changes in serum progesterone and estradiol between days 1-8 postpartum are represented in Table 1. Serum progesterone rose steadily between days 1 and 5 postpartum before reaching constant, elevated levels between days 5 and 7 before subsequently declining to intermediate levels on day 8. In contrast, serum estradiol levels decreased between days 1-3 postpartum before demonstrating a midluteal elevation between days 5 and 6 in association with the expected time of blastocyst implantation. Serum estradiol subsequently fell to basal levels between days 7 and 8. DISCUSSION The results of the present studies indicate that the endometrial changes associated with cellular growth and division in the postpartum, pregnant guinea pig occw in close as- 43 POSTPARTUM UTERINE REGENERATION TABLE 1. Changes in serum progesterone and estradiol levels between days 1 and 8 in the postpartum, pregnant guinea pig 1 Progesterone (ng/ml) Estradiol (pg/ml) 2 6.8 k1 16.9 kz 1.2 2.8 (9) (20) 13.1 k3 12.5 k3 1.6 1.6 (5) (4) All values are represented as group means 3: P 4 0.05; 1 vs 3: P < 0.01. 3 Dav Dostuartum 5 15.6 kz 3.1 (16) 2.2 f 1 0.3 (7) 25.6 k3 3.1 (10) 18.6 k 3 5.5 (4) 6 7 8 22.4 k3 2.2 (17) 15.8 k3 4.0 (5) 22.8 k 3 4.7 (18) 5.2 +' 1.5 (4) 15.7 k2 1.3 (11) 6.7 +' 0.9 (9) + SEM for (N) animals. Intergroup differences are denoted by 1 vs 2 or 2 YS sociation with the concomitant changes in circulating progesterone and estradiol levels. The pattern of mitotic activity exhibited by the glandular epithelium paralleled the temporal changes in serum estradiol levels. In a similar manner, stromal cell multiplication followed (approximately 24-48 hr) the temporal elevations observed in serum progesterone levels, especially between days 3 and 7 postpartum. Myometrial cell mitosis did not demonstrate any rate changes representative of the fluctuations in serum ovarian steroid hormone levels. The pattern of mitotic activity in the epithelial cells appeared to vary with the changes in both estradiol and progesterone levels. The day 1-2 decline in the epithelial cell mitotic index followed the mild decline in serum estradiol levels, during which time serum progesterone concentrations increased. The elevated epithelial mitotic activity between days 3-5 postpartum preceeded the subsequent elevation in serum estradiol levels between days 5 and 6, and declined to basal rates between days 6-8 as serum estradiol levels declined and progesterone concentrations rose. Thus, the temporal correlations observed in the cellular mitotic activity and serum steroid levels suggest that while the glandular epithelial and stromal changes are apparently modulated by estradiol and progesterone, respectively, the luminal epithelium changes occur relative to the fluctuating ratio of estradiol-to-progesterone in circulation. The results of the present study are in close agreement with respect to the site of action of estradiol and progesterone in the guinea pig uterus (Everett, 1962) and the effects of each relative to the cellular mitotic activity (Marcus, 1974a; Mehrotra and Finn, 1974). Of interest is the observation that in the postpartum guinea pig uterus, the uterine hyperemia which occurs in association with blastocyst implantation between days 5 and 7 (Garris and Whitehead, 1981; Garris, 1984a) is accompanied by a rapid rise in stromal cell mitotic activity. The concomitant elevation in both serum progesterone and estradiol levels during this time suggest that the hormonal modulation of the endometrial vascular response (Garris and Whitehead, 1981)and mitotic index are both causal and interdependent events. Similar correlations have been reported for the mouse (Finn, 1977) and rat (Marcus, 1974b; Krueger et al., 1975; Kang et al., 1975). These studies suggest that the pattern of endometrial cell mitosis in the postpartum guinea pig is very similar to that observed in the virgin, nongravid condition (Marcus, 1974a; Mehrotra and Finn, 1974).The ability to mimic the cyclic pattern of mitotic activity in the endometrial layers of ovariectomized guinea pigs with ovarian steroids (Marcus, 1974a) indicates that the temporal aspects of uterine mitotic activity observed in the present study were a result of the relative fluctuations in serum progesterone and estradiol levels. Considering both the rapidity of the endometrial changes that occur postpartum and the associated response elicited from the endometrial vascular bed by ovarian steroids (Garris and Whitehead, 1981), it is concluded that the guinea pig uterus is capable of returning to a near normal endometrial condition that is acceptable for successful nidation within 5 to 6 days postpartum. This is supported by the previous observations that blastocyst implantation is successful following postpartum breeding in the guinea pig (Garris and Whitehead, 1981) and that various other uterine parameters which are recognized to participate in the nidatory process also occur in association with endometrial reorganization in this species (Garris, 198413). These changes are apparently under the di- 44 D.R. GARRIS rect influence of the combined actions of progesterone and estradiol. LITERATURE CITED Everett, J. (1962) The influence of oestriol and progesterone on the endometrium of the guinea-pig in vitro. J. Endocrinol., 24:491-496. Finn, C.A. (1977)The implantation reaction. In: Biology of the Uterus. R.M. Wynn, ed. Plenum Press, New York, p. 245. Garris, D.R. (1984a) Ultrastructural aspects of the appositional stage of blastocyst implantation. Gynecol. Obstet. Invest., 17:lO-17. Garris, D.R. (1984b) Uterine blood flow, pH and pCOz during nidation in the guinea pig: Ovarian regulation. Endocrinology, 124:1219-1224. Garris, D.R., and D.S. Whitehead (1981) Uterine blood flow and timing of blastocyst implantation in the guinea pig. Am. J. Physiol., 241:E142-E145. Gibori, G., E. Antczak, and I. Rothchild (1977) The role of estrogen in the regulation of luteal progesterone secretion in the rat after day 12 of pregnancy. Endocrinology, 100:1483-1489. Kang, Y.-H., W.A. Anderson, and E.R. DeSombre (1975) Modulation of uterine morphology and growth by estradiol-170 and an estrogen antagonist. J. Cell Biol., 64:682-691. Korenman, S.G., R.H. Stevens, L.A. Carpenter, M. Robb, G.D. Niswender, and B.M. Sherman (1974) Estradiol radioirnmunoassay without chromatography: Procedure, validation and normal values. J. Clin. Endocrinol. Metab., 38t718-720. Krueger, W.A., W.J. Bo, and P.C. Hoopes (1975) A circadian rhythm of mitotic activity in the uterine luminal epithelium of the rat: Effect of estrogen. Anat. Rec., 183.563-566. Marcus, G.J. (1974a) Hormonal control of proliferation in the guinea-pig uterus. J. Endocrinol., 63:89-97. Marcus, G.J. (1974b)Mitosis in the rat uterus during the estrous cycle, early pregnancy and early pseudopregnancy. Biol. Reprod., 10:447-452. Mehrotra, S.N., and C.A. Finn (1974) Cell proliferation in the uterus of the guinea-pig. J. Reprod. Fertil., 37:405-409. Psychoyos, A. (1973) Hormonal control of ovo-implantation. Vitam. Horrn.. 31 :201-256.