Bovine uterine, cervical and ovarian estrogen and progesterone receptor concentrations

Bovine uterine, cervical and ovarian estrogen and progesterone receptor concentrations
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  Animal Reproduction Scrcttce, 16 ( 199 I ) 6 -7 Elsevier Science zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK ublishers B.V., Amsterdam 61 Bovine uterine, cervical and ovarian estrogen and progesterone receptor concentrations M. Vesanen”*‘, V. Isomaa”, M. Alankob and R. Vi&o’ Departmettt afChtical Chemrstry, Uniwrsity of 0th. SF-90220 Otdu, Finland bDepartment offobstetrics and Gynnecology, College of Veterinary Medicine. SF-04840 Hautj~rvi. Finland (Accepted28 March 1991) zyxwvutsrqponmlkjihgfedc ABSTRACT Vesanen. M.. Isomaa. V.. Alanko. M. and Vihko, R., 1991. Bovine uterine, cervica: and ovarian estro- gen and proge~leronc eceptor oncentrations. nim. Repfad. Sci., ZL: 6 zyxwvutsrqponmlkjih -7 I. Bovine uterine cytosol estrogen ERC) and progesterone receptors (PRC) were measured simul- taneously in the cndometrium and myometrium of cows with various serum pmgesterone concentra- tions. When serum prqesterone concentrations were low (less than 2.7 nmol I-‘), PRC concentra- tions in the endumetrium i 162 fmol mg- ’ cytosol rotein ) and in the myometrium ( 13 I5 mol mg-’ cymsoi proein) were relatively high. In animals with serum progeslerone concentrations be- wccn 2.7 and 6.4 nmol I-‘, endometrial and myomnrial PRC concentrations were 695 fmol mg-’ zyxwvutsrqp protein and 694 fmol mg- ’ protein, respectively. When the progestecnneconcentralion was above 6.4 nmol I- ‘. PRCcontcnts furtherdecreased in cndomctrium and myometrium (491 fmol mg-’ protein and 485 fmol mg-’ protein. respectively). ERC concentrations seemed to follow those of PRC, but mostly the changes were not statistically signiticant. No differences were observed in PRC or ERC concentrations between the endometrinm and myometrium at specilied serum progesterone leveis. Cervical PRC and ERC concentrations seemed to be lower than the corresponding uterine receptor levelsand in thecervix. too, reduced PRC concentrations wereassociated withendogenous progester- one production. The lauer tinding strongly suggests hat bovine uterine cervix tissue is under regula- rion by sex steroids. No differences in ERC or PRC contents were found between the two uterine horns of individual animals. Ovarian ERC and PRC concentrations were low compared to those de- teaed in the uwrus. Some synthetic CZI-pro&wins were bund more avidly by the bovine PRC than by that of human, sheep, rabbit and guinea pig. INTRODUCTION The mammalian uterus is highly sensitive to the ovarian steroid hormones estradiol and progesterone. It is generally accepted that steroid hormones me- diate their action via intracellular receptors. The concentrations of estrogen and progesterone receptors undergo characteristic variations throu&hout the -- ‘Author IO whom correspondence should be addressed. 0378-4320/91/503.50 0 1991 Elsevier Science Publishers B.V. All rights reserved.  reproductive cycle in response to changes in circulating sitAd hormone con- centrations. Progesterone and estradiol exert opposing effects on female sex steroid receptors in the mammalian uterus. Estradiol increases estrogen and progesterone receptor concentrations, while progesterone decreases both es- trogen and progesterone receptors by mechanisms which are not well under- stood (for reviews, see Katzenellenbogen, 1980; Leavitt et al., 1983; Clark et al., 1985). In a previous study, we characterized cytosol estrogen (ERC) and proges- terone receptor (PRC) concentrations in non-pregnant bovine endometrium at various physiological stages (Vesanen et al., 1988). The highest receptor concentrations were observed at postpartum anestrus and during the follicu- far phase of the estrous cycle, when progesterone production was low. During endogenous progesterone production by the corpus luteum in the luteal phase of the estrous cycle, ERC and PRC concentrations declined in the endometrium. To further study the hormonal regulation of bovine estrogen and progester- one receptors, we measured receptor concentrations simultaneously in theen- dometrium, myometrium, both uterine horns and in the cervix of animals having different serum concentrations of progesterone. n addition, we mea- sured ovarian receptor concentrations to characterize the role of circulating steroid hormones in the regulation of ovarian function. zyxwvutsrqponmlkjihgfed MATERIALS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH ND METHODS Animals and collection of samples The animals were Finnish dairy cows delivered to slaughterhouses. They were examined by rectal palpation of the genitalia to exclude those which had abnormalities in reproductive function. Blood samples were collected from jugular veins without anticoagulant. The uterus and ovaries were removed after slaughter and examined macroscopically. Tissue specimens (walls of the uterine horns, portio vaginalis cervicis and ovaries) were packed in foil, fro- zyxwvutsrq zen within 15 min of stunning and stored in liquid nitrogen for receptor anal- yses. Serum was separated from blood samples by centrifugation and stored at -20°C for steroid hormone assays. Measurement 9 f cpod rsrrogen and progesterone receptor concentrations In this paper, cytosol receptors (ERC; PRC) refer to those found in the cytosot fraction following homogenization of the tissue in buffer and ultra- centrifugation of the homogenate. Endometrium and myometrium were separated from frozen tissue samples using a scalpel. Endometrial specimens were scraped from surface of the en-  BOVINE ESTROGEN AND PRDCESTERONEREC EI’TOP 63 dometrium with a scalpel. Myometrial samples were cut from middle stratum of the uterine wall. Macroscopically selected stromal tissue was used in ovar- ian steroid receptor assays. The corpus luteum, follicles with their walls and the ovarian capsule were removed from specimens before assay. Measure- ment of ERC and PRC concentrations was performed as described previously (Vesanen et al., 1988). Briefly, tissue pieces were wei@ted, minced and ho- mogenized in TETMG 10 G buffer at +4”C. The homogenate was centri- fuged and supematants saved for receptor and protein assays. In the ERC assay, the cytosol was incubated with various concentrations (0.3-10 nmol I-‘) of (3H)estradiol. Non-labeled testosterone was used to eliminate possi- ble interference by serum proteins. Non-specific binding of t3H)estradiol was estimated by using a 200-fold molar excess of non-labeled estradiol. After in- cubation, non-bound steroids were separated by dextran-coated charcoal and radioactivity was counted in a liquid scintillation counter. In the assay of PRC, cytosol was incubated with various concentrations (0.4-14 nmol 1-l) of (3H)ORG 2058. To estimate non-specific binding of t3H)DRG 2058, a 200- fold molar excess of non-labeled ORG 2058 was used. The method of Scatchard ( 1949) was used to calculate the binding data, corrected for non-specific binding, for both cytosol receptors. A control cy- tosol sample was used in every measurement to monitor assay performance. The receptor concentrations were expressed as fmol mg-’ cytosol protein. The concentrations of cytosol protein were measured according to Bradford ( 1976) using BIO-RAD protein assay reagents ( Bio-Rad Laboratories Rich- mond, CA). Measurement of the relative binding affinities of various steroid hormones and other compounds toward ERC and PRC was performed using constant molar concentrations of labeled tracer ( t3H)E2 or (‘H)ORG 2058) and var- ious quantities of non-labeled competitors in incubations. Relative binding affinities of the compounds were estimated from the amounts of ligands re- quired to decrease the binding of the tracers by 50%. Measurement zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON f serum sreroid hormone concentrations The serum concentrations of progesterone were measured by radioimmu- noassays after solvent extraction and chromatographic purification of the samples as described previously ( Apter et al., 1976; Hammond et al., 1978 . Briefly, unconjugated steroids were extracted from serum and then fraction- ated on Lipidex-5WOTM microcolumns (Packard-Becker, B.V., Chemical Operations, Netherlands), followed by radioimmunoassay of progeatetone from the appropriate fraction using a specific antiserum (Jlnne et al., 1974; Apter et al., 1976).  64 M. VESANLN ET AL. Other methods Statistical comparisons of the results were performed by one-way analysis of variance and t-statistics by the Bonferroni method (Wallenstein et al., 1980). Linear regression analysis was used to evaluate the correlations. RESULTS Since no information was available concerning the stability of bovine PRC and ERC in vitro, we studied the effects of tissue processing on receptor con- centrations. Tissue samples were usually frozen within I5 min of stunning, but storage of tissue at room temperature for up to 1 h did not measurably affect receptor levels. In addition, storage of uterine tissue or cytosol at - 70” C for 8 months did not decrease receptor concentrations. In estrogen receptor assays, maximum specific binding was obtained using a first incubation at + 4”C, followed by 1 h at + 30°C. In the case of progestin receptors, incuba- tions were carried out at +4”C, the binding reached a maximum by 2 h and it remained stable for at least 24 h (data not shown). Bovine uterine cytosol ERC and PRC concentrations were measured si- multaneously in endometrium and myometrium from animals with various serum progesterone evels. The animals were divided into three groups. Group I (low progesterone) consisted of animals with serum progesterone concen- trations below 2.7 nmol I-‘, which carresponds o a progesterone concentra- tion of 10 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA mol 1-l in residual milk, the limit used in our previous study (Vesanen et al., 1988). The second group (medium progesterone) comprised animals with serum progesterone from 2.7 to 6.4 nmol I-‘, and the third group (high progesterone) consisted of animals with serum progesterone concen- trations above 6.4 nmol 1- ‘. At low serum progesterone levels, ERC contents tended to be higher in the endometrium ( 29 12 257 (mean -C tandard deviation (SD) ) fmol mg- ’ cy- tosol protein, n-24) and myometrium (237 I 164 fmol zyxwvutsrqponmlkjih g-’ ) than at me- dium ( 164 + 92 fmol mg- ’ in endometrium, 135 t 99 fmol rng ’ in myomet- rium, )I= 12) and high levels of progesterone ( 173 2 63 fmol mg-’ in endometrium. 132 2 59 fmol mg-’ in myometrium, rr= IO) (Fig. I ). These changes were, however, not statistically significant. ERC concentrations in the endometrium and myometrium correlated significantly (PC: 0.00 1) in in- dividual animals. The same .&ding was observed concerning the PRC con- tents of endometrium and myometrium (Pz 0.001, Fig. 2 ). PRC levels were relatively high both in endometrium ( 11625692, fmol mg-‘. n-24) and myometrium (13152 136 fmol mg-‘) when serum pro- gesterone concentrations were low (less than 2.7 nmol I- ’ ). PRC concentra- tions were lower in both the endometrium (@Sk 343 fmol mg- ‘. n= 12) and myometrium (694 + 362 fmol mg- ’ ) when serum progesterone concentra-  65 iYT w .6,4 Fig. I. Bovine uterine ERC concentrations in endometrium (E) and myometrium (M). Ani- mals were divided into three groups according to their serum progesterone concentration: pru- gcsterone less than 2.7, 2.7-6.4 and greater than 6.4 nmol I-‘. The number of animals in each group was 24. I2 and IO, respectively. Values are expressed as the means?SD. zyxwvutsrqponmlkji 1000 soa Fig. 2. Bovine uterine PRC concentrations in endometrium (E) and myometrium (M). For other details. see the legend IO Fig. I Signiiicartces: zyxwvutsrqponmlkjihgfedcbaZYXWV P-c 0.05; -PC 0.01 “PC 0.00 I. tions were at a medium level (2.7-6.4 nmol I-‘), and PRC concentrations were lowest in the endometrium (49 12 23 1 mol mg-I, PI= 10) and myomet- rium (485 5244 fmol zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON g-’ ) when serum progesterone levels were high
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