Cyclic Adenosine Monophosphate Regulation in the Rabbit Corpus

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regulation by LH/cyclic AMP. Progesterone production by corpus luteum (CL) incubated with vehicle, 3-isobutyl-l-methyl-xanthine (IBMX),. hCG, or hCG + IBMX ...
BIOLOGY OF REPRODUCTION 53, 718-723 (1995)

Estrogen Uncouples Steroidogenesis from 3',5'-Cyclic Adenosine Monophosphate Regulation in the Rabbit Corpus Luteum' David H. Townson, 2 '3 P. Landis Keyes,3 and Jack L. Kostyo 4 Departmentof Physiology,3 The Reproductive Sciences Program, and the Michigan DiabetesResearch and Training Center,4 The University of Michigan Medical School Ann Arbor, Michigan ABSTRACT The hypothesis was investigated that estradiol, the luteotrophic hormone inthe rabbit, uncouples luteal progesterone production from regulation by LH/cyclic AMP. Progesterone production by corpus luteum (CL) incubated with vehicle, 3-isobutyl-l-methyl-xanthine (IBMX), hCG, or hCG +IBMX was compared inpseudopregnant rabbits treated continuously with estradiol (estradiol-maintained),withdrawn from estradiol for 24-48 h (estradiol-withdrawn), or withdrawn and then replaced with estradiol for 6 or 24 h (estradiol-replaced). Progesterone production inestradiol-maintained rabbits was not altered by hCG and/or IBMX, but was stimulated significantly inestradiol-withdrawn rabbits. This response was reversed (i.e., abolished) in estradiol-replaced (24 h) rabbits. The loss of responsiveness to hCG was not attributable to impaired accumulation of cyclic AMP: basal and hCG-stimulated cyclic AMP concentrations were similar in luteal tissues of estradiol-maintained and estradiol-withdrawn rabbits. The loss of responsiveness to hCG was also not a consequence of maximal progesterone production: CL of estradiol-replaced (6 h) rabbits were also insensitive to hCG, and this occurred before progesterone production attained a maximal rate. We conclude that astriking feature of the luteotrophic action of estrogen isto uncouple the regulation of progesterone production from cyclic AMR

INTRODUCTION

conditions in which estrogen is present or acutely withdrawn [8, 9].

A characteristic of rabbit luteal tissue is the capacity for sustained synthesis of progesterone in incubations [1] or in organ cultures [2] in the absence of hormones added extrinsically. Recently, we reported that progesterone synthesis in rabbit luteal tissue is insensitive to stimulation by either hCG or cyclic AMP, in contrast to Graafian follicles, which exhibited pronounced responses to hCG or cyclic AMP [31. Similarly, Dorrington and Kilpatrick [4] found that progestin synthesis in rabbit corpus luteum (CL) was stimulated only to a slight extent by LH and cyclic AMP when compared with the substantial response of ovarian interstitial tissue. From these observations, we have proposed that steroidogenesis in the rabbit CL is not regulated by cyclic AMP, as is the case for ovarian follicles [5, 61, but rather that progesterone is produced autonomously [3]. Further, as a working hypothesis, we proposed that the role of estrogen, which is considered the luteotrophic hormone in this species [7], is to maintain the intracellular systems that enable progesterone to be produced autonomously [3]. In the present work, the hypothesized role of estrogen was tested more directly by determining the capacity of rabbit luteal tissue to respond to hCG or cyclic AMP under

MATERIALS AND METHODS Materials Equine CG (eCG) was obtained from Diosynth (Chicago, IL), and hCG for injection was obtained from Sigma Chemical Co. (St. Louis, MO). Human CG for use in short-term incubations was from NIH (CR-127). Medium 199 (M199; with Earle's salts and L-glutamine) was from GIBCO (Grand Island, NY); to it was added 2.2 g/L of sodium bicarbonate (Fisher Scientific, Springfield, NJ), 50 mg/ml gentamicin sulfate (Sigma), and 1% BSA (Armour Pharmaceutical Co., Kankakee, IL). N6-monobutyryladenosine 3',5'-cyclic monophosphate (cyclic AMP) and 3-isobutyl-l-methyl-xanthine (IBMX) were from Sigma Chemical Co. Methods Pseudopregnancy was induced in New Zealand White rabbits (3.0-4.5 kg) by i.m. injection of 40 IU eCG followed 50 h later by i.v. injection of 40 IU hCG. The day of hCG injection (day of ovulation) was designated as Day 0. On Day 1 of pseudopregnancy, all rabbits received two polydimethylsiloxane implants (1.2 cm, filled length) containing crystalline estradiol-173 s.c. as described previously [8, 91. Estrogen exposure was manipulated in vivo by removal or reinsertion of the implants during the time periods indicated in each experiment. At the end of the experiment, each rab-

Accepted May 5, 1995. Received February 24, 1995. 'Supported by NIH Grants HD28396 (P.L.K.), T32-HD07048 (D.H.T.), and P30 HD18258 Assay and Reagents Core Facility. This research was presented in part at the 76th Annual Meeting of The Endocrine Society, Anaheim, CA, June 1994 (Abstract 216). 2Correspondence: D.H. Townson, Department of Physiology, 7627 Medical Science II, The University of Michigan, Ann Arbor, MI 48109-0622. FAX: (313) 936-8813.

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bit was anesthetized by an i.v. injection of 195 mg of sodium pentobarbital. The ovaries were then removed and placed in ice-cold M199 for subsequent dissection of CL. Short-Term Incubation of CL for Measurement of ProgesteroneProduction Ovaries from each rabbit were kept separate, and the CL were dissected with magnification provided by a dissecting microscope. The CL were teased open to facilitate exposure of the tissue to the incubation medium. Each CL was incubated with gentle shaking at 37 0C for 5 h (see individual experiments) in 15 X 85-mm stoppered glass culture tubes (1 CL per tube) containing 1.0 ml M199 with 1% BSA, with or without treatments (hCG, IBMX, or cyclic AMP; see individual experiments), in an atmosphere of 95% 02:5% CO 2. Usually 10-12 CL were obtained from each rabbit; therefore, each treatment was run in duplicate. A single observation (n = 1) constitutes the mean of duplicate determinations. At the end of the incubation period, the medium was removed and frozen for later assay of progesterone. The CL were placed on filter paper, blotted to remove excess liquid, and then weighed. Progesterone in the incubation medium was measured directly without extraction as described previously [1]. The 50% binding point for the assay was 2.7 ng/ml. Intra- and interassay coefficients of variation were 6.3% and 11.9%, respectively. M199 with 1% BSA had negligible inhibiting activity (< 40 pg/ml) in the assay. The progesterone assays were conducted in the Chemistry Core of the Michigan Diabetes Research and Training Center. The assay results are expressed as nanograms of progesterone released into the medium per milligram of tissue wet weight. Incubationsfor Measurement of Cyclic AMP Content While the rabbit was anesthetized, the CL were dissected rapidly from the ovary in situ, and each CL was gently teased open with fine forceps and either frozen immediately (to determine basal cyclic AMP concentrations) or incubated for 15 min in prewarmed M199-1% BSA containing hCG (0, 0.1, 1.0, 10 pg/ml) and IBMX (5 mM). At the end of the incubation period, the CL were placed on filter paper, blotted lightly, and then frozen with aluminum tongs cooled to the temperature of liquid nitrogen. The frozen tissue was then weighed, placed into 2.0 ml of ice-cold 10% trichloroacetic acid (TCA), and homogenized in a glass/glass homogenizer. The homogenate was centrifuged for 10 min, and a 1.5-ml aliquot of the supernatant was extracted 4 times with 3 ml of water-saturated ether to remove the TCA. Residual ether was driven off by heating the extract to 50°C. The extract was then frozen in liquid nitrogen and stored at - 20C until assayed for cyclic AMP by RIA. The assay was conducted in the Chemistry Core of the Michigan Diabetes

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FIG. 1. Invitro effects of hCG (10 lg/ml) in absence or presence of IBMX (5 mM) on progesterone production during 5-h incubation period by CL isolated from ovaries of Day-11 pseudopregnant rabbits treated continuously with estradiol (top panel) or treated with estradiol for 9 days and then removed from estradiol treatment for 2 days (bottom panel). Values are means SEM of four separate experiments. *Significant differences from control (p < 0.05).

Research and Training Center with use of the double antibody procedure of Vaitukaitis et al. [10]. The results are expressed as femtomoles of cyclic AMP per microgram of tissue protein. ProteinAssay Total protein content of homogenates of CL was measured according to the modified Lowry procedure described by Peterson [11]. StatisticalAnalyses Data were analyzed by two-way analysis of variance in which rabbit and treatment effects were partitioned. Significant treatment effects (p < 0.05) were further analyzed by comparing means of treatments using a Student-NewmanKeuls Multiple Range test. Comparisons between estrogen treatment groups were conducted by means of Student's ttest. No pooling of rabbit CL was necessary to complete the 4-5 treatments (in duplicate) of a given experiment. Therefore, rabbit is defined as the experimental unit, and the number of rabbits used for each experiment is designated in the figure legends.

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