Direct effect of estradiol-17 on progesterone accumulation by ovarian granulosa cells from rhesus monkeys.код для вставкиСкачать
American Journal of Primatology 17537-92 (1989) Direct Effect of Estradiol-17p on Progesterone Accumulation by Ovarian Granulosa Cells From Rhesus Monkeys REINHOLD J. HUTZ1, PATRICIA M. MORGAN', GAIL S. KRUEGER', MAUREEN DURNING2, AND DONALD J. DIERSCHKE2 'Department of Biological Sciences, University of Wisconsin-Milwaukee; 2Wisconsin Regional Primate Research Center, Madison We have previously demonstrated that estradiol-17P (E,) administered in vivo induces atresia of the dominant ovarian follicle (DF). Whether this effect is exerted directly a t the ovarian level or by central mediation has not been confirmed. The present study was designed to assess whether E2 in amounts similar to those found in monkey follicular fluid (FF) directly alters in vitro progesterone (P) accumulation by granulosa cells (GC) aspirated from follicles in cycling rhesus monkeys. Follicular contents were aspirated from three to five animals on each of days 8-13 of the cycle. GC were plated at a density of 50,000 viable GU0.5 ml medium; GC were incubated with 0 or 2-2,000 ng/ml E2, and cultures were maintained for 72 h. P accumulation by GC collected on day 8 and treated with 2 ng/ml Ez was augmented 37.5 -+ 5.5% (X S.E.M.; P < .05) over controls but was diminished significantly a t 20 ng/ml(-55 2 18%with respect to controls), 200 ng/ml (-73.7 k 13.2%),and 2,000 ng/ml (-77.3 k 18.4%).A similar dose-response relationship was noted on other cycle days. At a concentration of 2,000 ng/ml, Ez significantly reduced P 91.5 2 8.5% (day 10),81.5 18.5% (day ll),84.3 i 4.7% (day 12), and 53.7 t 15.8% (day 13). We conclude that Ez at concentrations found in FF can inhibit P output by monkey GC through a direct action. * * Key words: estrogen, follicle cells, in vitro, Macaca mulatta INTRODUCTION We have previously demonstrated that treatment with estradiol-17P (E,) induces atresia of the dominant ovarian follicle (DF) in rhesus monkeys, resulting in extension of the follicular phase by 8-10 days and ovulation of a substitute follicle [Dierschke et al., 19851; biochemical and morphologic changes in the Received for publication August 4,1988;revision accepted October 10,1988. Address reprint requests to Dr. Reinhold J. Hutz, Department of Biological Sciences, University of Wisconsin-Milwaukee, P.O. Box 413,Milwaukee, WI 53201. 0 1989 Alan R. Liss, Inc. 88 / Hutz et al. intrafollicular milieu indicative of atresia characterize our model [Hutz et al., 19861. The atretogenic effects of Ez may be partially mediated by alterations in follicle-stimulating hormone (FSH) secretion [Zeleznik, 1981; Dierschke et al., 19851; however, Ez also exerts a direct effect on the monkey DF as evident from local application of Ez [Hutz et al., 19881. Thus, the site of action for E2 is not firmly established. The present study is the first designed to assess whether Ez, in concentrations similar to those found in monkey follicular fluid (FF), directly alters in vitro progesterone (PI accumulation by granulosa cells (GC) aspirated from cycling rhesus monkeys. MATERIALS AND METHODS Animals Twenty-three mature rhesus monkeys (Macacu mulattu) exhibiting normal menstrual cycles (24-32 days in length, and >lO-day luteal phases, based upon perineal sex skin [Czaja et al., 19751)were used. Care and housing of animals and laparoscopic procedures were as previously described [Hutz et al., 19851. The DF was identified, and size was estimated a t laparoscopy [Clark et al., 19791 on day 6 or by ultrasonography on days 8-10 [Morgan et al., 19871 of the menstrual cycle; day 1of the cycle was the first day of menstrual bleeding. Aspiration and In Vitro Culture Contents of the DF present on either days 8,9,10,11,12, or 13 were aspirated as previously reported [Hutz et al., 19861. Follicle contents were analyzed from three t o five animals per cycle day. Briefly, aspiration was performed under sterile conditions utilizing a steel 2-inch, 25-gauge needle attached to a 1-ml syringe. The aspiration medium contained Dulbecco’s modified Eagle’s medium mixed 1:l with Ham’s F-12 medium [Sanders & Midgley, 19831, supplemented with 100 ng/ml human FSH (hFSH), 100 pg/ml gentamycin, 12.5 mM HEPES buffer, and 1 U/ml heparin. FF was centrifuged a t 500g for 10 min, the supernatant was discarded, and the cellular pellet was recovered. The number of GC in aspirates was determined using a hemocytometer and averaged 8.46 k 1.11 x 105(23) (X & 1 S.E.M. [nl); range, 1.83-19.82 x lo5). GC viability was estimated with 0.2% trypan blue, and cells were resuspended and seeded at a density of lo5 viable GC per well in 0.5 ml medium in 8-chamber Lab Tek culture slides (Miles Labs, Naperville, IL). Mean GC viability was 54.0 5 3.0 (23); (range, 16-77%), and did not change with cycle day. GC were incubated in duplicate cultures with either 0 , or 2, 20, 200, or 2,000 ng Ez/ml medium for 72 h in 5% C02 in air with high humidity (>go%). The amounts of Ez used represented concentrations known to be present in rhesus monkey ovarian FF a t day 10 of the cycle [Hutz et al., 19861. Culture medium was removed after the incubation period and assayed for P, since production of this steroid is an excellent indicator of GC viability [Bomsel-Helmreich et al., 1979; McNatty et al., 19791. Estimation of steroid accumulation in culture was expressed in terms of unit steroid10.5 ml culture medium (containing lo5 viable GC at the start of culture)/72 h. Radioimmunoassays Concentrations of luteinizing hormone (LH) in serum and P in FF culture medium were determined according t o methods previously described [Clark et al., 1978,1979; Hutz et al., 19861. Assay sensitivity was 100 ngiml (LH) and 0.5 ngiml In Vitro Effects of Ez on Granulosa Cells / 89 TABLE I. Accumulation of Progesterone by Granulosa Cells In Vitrot Estradiol-178 concentration (ng/ml) Day of cycle 8 9 10 11 12 13 20 2 * +37.5 5.5* N.D. + 132.9 - 18.0 N.D.* + 0.5 + 112.4 -55.0 * 18.0** N.D. 2,000 200 -73.7 2 13.2** N.D. + 56.0 -46.0 -22.0 -84.5 i 15.5* + 5.3 +34.0 i 1.0* -16.3 +0.3 i 1.7** ? -77.3 i 18.4** N.D. -91.5 8.5** * 10.0* N.D.* -81.5 ? 18.5* -84.3 i 4.7 -53.7 5 15.8*** N.D. tEffect of estradiol-17P. Values are percentages with respect to control incubations N.D. = Not detectable in assay; equivalent to -100%. *,**,***Differenceswithin rows denote significance (P < ,051. f 1 S.E.M. (P).Intra- and interassay coefficients of variation were <5% and <lo% for LH and <lo% and <17% for P, respectively. Statistical Analyses GC from one animal were divided into control and treatment groups (a statistical block), although this was not always possible when too few cells were available. In the latter case, only means of pooled data from several animals within a treatment are presented in Table I. In some instances, results were dichotomous at low Ez concentrations; to demonstrate this more clearly, we partitioned these data into two subgroups based upon statistical differences between the subgroups. Each value in Table I is a mean of two to three separate animal replicates, with each replicate consisting of at least three GC incubations. Data were transformed (arcsin & log x) and analyzed by one-way analysis of variance followed by Student-Newman-Keuls test, or by Kruskal-Wallis test for nonparametric data. Values are not shown in Table I when these were less than the sensitivity of the assay (not detectable); these values were taken as a 100% reduction in P accumulation. Because of interanimal variation, data are presented as percent of control (0 ng/ml E2). P < .05 was considered significant. RESULTS In general, there was a differential, sometimes dichotomous effect of Ez upon P accumulation by monkey GC in vitro with Ez concentrations to 20 ng/ml; P amounts significantly decreased at 200 ng/ml on five of six'cycle days (days 8-12) (Table I). Specifically, 2 ng/ml EZ augmented P output by GC aspirated on day 8 vs. untreated control incubations. At days 9 and 11, P accumulation in all treated incubations, regardless of dose, fell significantly below that of controls. At days 10 and 13, effects of E2 were both positive and negative to 20 ng/ml; P accumulation reached control levels at 200 ng E2/ml (day 13) and was significantly depressed at 2,000 ng Ez/ml on both days 10 and 13. GC aspirated on day 12 showed reduced amounts of P at 2,000 ng/ml Ez. When aspiration was performed prior to the LH surge (e.g., days 8, 9, ll),P accumulation in vitro increased relative to control, regardless of treatment (data not shown). When aspiration occurred on the day of or after the LH surge (days 12 90 I Hutz et al. and 13), there was less of an apparent diminution in P accumulation at 200 and 2,000 ng E2/ml; this was, however, not significant. DISCUSSION The present study is the first to show that E2 a t concentrations similar to those found in FF (-1,000 ng E2/ml) [Hutz et al., 19861, can directly inhibit accumulation of P by GC aspirated from follicles in cycling rhesus monkeys. The differential response t o varying E2 concentrations was dose dependent: lower concentrations enhanced P production, whereas higher concentrations were suppressive. These dose-dependent estrogen responses have also been shown with regard to folliculogenesis in rodents [Kim et al., 19841. The apparent biphasic actions of low-dose E2 on GC P production may be attributed both to small sample size and to a real dichotomy also seen in our previous studies in which rhesus monkeys received E2 systemically [Dierschke et al., 19851. The apparent diminution in E2’sinhibitory effects on P production by GC from animals experiencing the endogenous surge of LH may well have been due to some luteinization of follicular cells and intrinsic production of P [Hutz et al., 1987al; however, the inhibitory effects of E2 were still evident regardless of whether GC were exposed to the LH peak. In previous work, we have demonstrated in rhesus monkeys that four Silastic capsules containing crystalline E2 placed subcutaneously (s.c.) on day 6 of that menstrual cycle for 24 h induced atresia of the DF in 100% of animals [Dierschke et al., 19851. The microenvironment of the DF was altered by this treatment such that FF amounts of E and P were reduced; the steroidogenic capacity of aspirated GC was attenuated in vitro, as was the viability of GC and oocyte [Hutz et al., 19861. Some of the atretogenic effects of E2 may be mediated via suppression of FSH [Zeleznik, 1981; Clark et al., 1981; Dierschke et al., 19851, although superimposition of exogenous gonadotropins upon E2 treatment did not overcome the atretogenic effect [Dierschke et al., 19861.In addition, direct injection of E2 into the stromal tissue surrounding the DF induced atresia with little perturbation in peripheral E and no discernible effect on gonadotropin secretion [Hutz et al., 19881. Using immature hamsters treated with pregnant mare’s serum gonadotropin to induce follicular development, we have also shown that diethylstilbestrol (DES) and E2 can inhibit accumulation of estrogen by GC directly in vitro [Hutz et al., 1987133 a t a locus other than the aromatase complex [Hutz et al., 1987~1;in this model and in cultures of whole follicle explants from cycling hamsters [Bejvan & Hutz, 19871,P accumulation was enhanced by coincubation with E2. In the present study, P accumulation by GC declined in coculture with E2. The discrepancy in P production with E2 may be due to species differences, particularly since the attenuated P concentrations also characterize granulosa cells and follicular fluid of rhesus monkeys administered E2 in vivo [Hutz et al., 19861. We do not presently know whether E2 has a direct effect upon aromatase enzymes in monkey GC; however, E2 may directly effect alterations in other steroidogenic enzymes, e.g., 3p-hydroxysteroid dehydrogenase and 17-a-hydroxylase [Munabi et al., 19831. We conclude that at “physiologic” concentrations approximating those found in dominant follicles from rhesus monkeys, E2 can reduce accumulation of P by dispersed GC owing to a direct effect in vitro. Collectively, this evidence strengthens our contention that E2can exert an effect at the level of the ovary. We are currently investigating the locus (loci) of this effect and mechanism(s) of action. In Vitro Effects of E2 on Granulosa Cells / 91 CONCLUSIONS 1. At concentrations approximating those found in rhesus monkey follicular fluid, estradiol-l7p can reduce accumulation of progesterone by granulosa cells by a direct effect in vitro. 2. Collectively, our evidence strongly suggests th a t Ez can exert a negative effect on folliculogenesis directly at the level of the ovary. ACKNOWLEDGMENTS The authors wish to thank Dr. S. Raiti of the National Hormone and Pituitary Program for supplying the hFSH, and Ms. Dorothy Perry and Ms. Alnita Allen for the expert preparation of this manuscript. Portions of this research were presented at the 11th Annual Meeting of the American Society of Primatologists, New Orleans, June 2-5, 1988. This work was supported by NIH Grants HD-17370 and RR-00167. This is publication 28-014 of the Wisconsin Regional Primate Research Center. This study was conducted in accordance with the “Guide for the Care and Use of Laboratory Animals,” NIH Publication No. 85-23 and Public Law 89-544, “The Animal Welfare Act,” August 24, 1966, and its Amendments. Patricia Morgan is now at the DeDartment of Physiology, University College Galway, Galway, Ireland. REFERENCES Bejvan, S.M.; Hutz, R.J. 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