Modulation of Ovarian Adenylate Cyclase Response to Gonadotropins Following Luteinization of the Rat Ovary C. Y. LEE* Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, 55455, and Department of Molecular Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901 an average 6-fold increase in enzyme activity. On the other hand, the adenylate cyclase of the luteinized ovaries lost its response to FSH. The hFSH-stimulated activity observed could be attributed to hLH contamination, since the apparent Km for hFSH was markedly higher (92 to 102 X 10~9M) than that

ABSTRACT. The hFSH- and hLH-sensitive adenylate cyclase activities of immature and luteinized rat ovaries were studied. Both immature and luteinized ovaries contain an enzyme activity highly responsive to gonadotropin stimulation. Satisfactory response of the ovarian adenylate cyclase activity obtained under optimal conditions required gentle homogenization of ovarian tissues. The enzyme activity of the immature ovaries was highly sensitive to hFSH (5- to 7-fold over the basal activity). The stimulation cannot be attributed to hLH contamination, since hFSH had a larger stimulatory effect than hLH in immature ovaries and the apparent Km for both hFSH and hLH were about the same. The adenylate cyclase of the heavily luteinized ovaries was found more responsive to hLH after luteinization by PMSG-hCG injection. hLH caused

G

ONADOTROPINS are known to initiate and control a number of biochemical events in the rat ovary. One of the early steps in gonadotropin action is the stimulation of the adenylate cyclase system, thereby increasing cyclic AMP formation. Marsh and associates (1,2) found that LH stimulated cyclic AMP accumulation in incubated slices of bovine corpus luteum and that addition of exogenous cyclic AMP to corpora luteal slices caused a significant stimulation of progesterone synthesis. Subsequently, the stimulation of cyclic AMP formation and of the adenylate cyclase activity by LH and FSH were also demonstrated in various ovarian structures of Received August 25, 1976. Supported by grants from USPHS (HD 10075 and HD 9140). Part of this work was presented at the 58th Annual Meeting of the Endocrine Society, San Francisco, California, June 23-25, 1976. * Present address: Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455.

for hLH (6.1 x 10- 9 M). The loss of FSH-stimulated

adenylate cyclase activity associated with the increase in LH-stimulated activity of the luteinized ovaries is consistent with the previous findings of disappearance of FSH receptors and increase in LH receptors following luteinization. These data indicate that modulation of the gonadotropin receptoradenylate cyclase system can be important in the regulation of ovarian response to gonadotropin stimulation. (Endocrinology 101: 876, 1977)

several species (3-12). The present report describes an adenylate cyclase system in the rat ovary that is highly responsive to gonadotropins and presents data concerning the modulation of the responsiveness of FSHand LH-stimulated adenylate cyclase to gonadotropin following luteinization. Materials and Methods Ovarian homogenates Immature female Holtzman rats were used at 22 to 34 days of age. Heavily luteinized ovaries were obtained from rats primed with 50 IU of PMSG at 25 days of age and 50 IU of hCG 56 h later. The animals were killed 10 days after PMSG. Ovaries were homogenized in 10 volumes (wt/vol) of 0.33M sucrose containing 1.0 mM EDTA and 10 mM Tris-HCl, pH 7.5. The homogenates were prepared using 6 strokes in a motor driven Teflon-glass homogenizer while keeping the homogenizer chilled in ice. The homogenates were filtered through four layers of cheesecloth and diluted to a final concentration of 40 mg wt/ml. Twenty-five /xl aliquots of homogenates were used for adenylate cyclase

876

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 23:43 For personal use only. No other uses without permission. . All rights reserved.

877

OVARIAN ADENYLATE CYCLASE ACTIVITY assay. In each experiment, the luteinized ovaries were obtained from 3 to 5 rats and the immature ovaries from at least 15 rats.

300

Adenylate cyclase assay

250

Enzyme activity was determined as described by Birnbaumer et al. (11) with a minor modification. The incubation mixture contained 25 mM Tris-HCl, pH 7.0, 5.0 mM MgCl2, 1 mM EDTA, 1 mM 3-isobutyl-l-methylxanthine, and an ATPregeneration system consisting of 20 mM creatine phosphate and 0.2 mg/ml of creatine kinase at pH 7.0, 1 mM cyclic AMP, 1 mM [a-32P]ATP, and hormone as indicated. The reaction was initiated by the addition of homogenates to give 1 mg wt per tube. The final volume of reaction was 50 fi\. Incubation was at 30 C for 20 min. The reaction was terminated by addition of 0.1 ml of stopping solution containing 40 mM ATP, 12.5 mM cyclic AMP, [3H]cyclic AMP (for estimating recovery), followed by heating at 100 C for 3 min. Cyclic AMP was isolated using Dowex and alumina chromatography (13). The radioactivity present in the eluate containing [32P]cyclic AMP and [3H]cyclic AMP was measured in a Beckman LS-100 C liquid scintillation counter. Enzyme activity was expressed as picomoles cyclic AMP formed per 20 min per mg protein. Protein was determined by the method of Lowry et al. (14). Bovine serum albumin (Fraction V) was used as a standard. Materials Highly purified hFSH (LER-1575C), highly purified hCG (CR121) and ovine prolactin-S6 were obtained from NIAMDD, NIH, Bethesda, Md. Highly purified hLH (22870-1B) and partially purified hFSH (R-21275) were obtained from Dr. R. J. Ryan of Mayo Clinic, Rochester, Minnesota. The contamination of hFSH (LER1575C) by hLH was 174 IU/mg by bioassay or approximately 9% by weight assuming a potency of 1800 IU/mg for pure hLH. Prostaglandin (PG) E, was kindly supplied by Dr. J. Pike, the Upjohn Co., Kalamazoo, Michigan.

hCG

en

\ c

200 5_ vt

2

4 o

150

50

< 4

XO

0

1

2 ATP(mM)

3

FIG. 1. Effect of ATP on basal and hCG-stimulated (10 /xg/ml) ovarian adenylate cyclase activities. Luteinized ovaries were removed after treatment of immature rats with PMSG-hCG (10 days after PMSG). Ovarian homogenates were prepared as described in Materials and Methods. Homogenates (79 jug protein per assay tube) were incubated with a constant concentration (5 mM) of Mg++ and the indicated concentrations of ATP under standard assay conditions, at 30 C for 20 min, as described in Materials and Methods. For each concentration of ATP used, the ratio of hCG-stimulated activity to the basal activity was calculated (A A). The symbols on the solid lines represent duplicate determinations of a single pool.

tion medium on basal (without hormone) and hCG-stimulated adenylate cyclase activities of the luteinized ovaries (PMSGhCG primed) is shown in Fig. 1. Both the basal and hCG-stimulated activities were enhanced by increasing concentrations of ATP from 0.25 to 1.0 mM. The hCG-stimuResults lated activity is increased more than the basal activity, as reflected by increasing horProperties of ovarian adenylate cyclase mone stimulation relative to basal. Optimum Under the standard assay conditions, the hormone stimulation (5.5-fold over basal) effect of ATP concentration in the incuba- was obtained at 2.0 mM ATP under the

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 23:43 For personal use only. No other uses without permission. . All rights reserved.

LEE

878

of the luteinized ovaries is illustrated in Fig. 2. At 1 mM ATP, maximal hCG stimulation (5.8-fold over basal) was obtained at a Mg ++ concentration of 3.0 mM as shown in the upper panel of Fig. 2. The same optimal assay conditions were found for the hCGand hFSH-stimulated adenylate cyclase in the immature rat ovaries. When the assay was carried out at 2.0 mM ATP (lower panel of Fig. 2), the ratio of enzyme activities was maximal at 4.0 mM Mg ++ with a 6.2-fold increase in hCG-stimulated activity relative to basal. The absolute response of the enzyme activity to hCG declined as the concentration of Mg ++ was increased above

600 c 500 ~ o

Endo • 1977 Vol 101 • No 3

I mM A T P

E \ 400

i o > 300

8 mM (lower panel). - 1.000

The time course of both basal and hCGstimulated adenylate cyclase activities of the luteinized ovaries is shown in Fig. 3. A lag phase was observed in the absence and presence of hCG for the first 4 min incubation. After this time the enzyme activity was linearly related to the incubation time up to 25 min.

2mM ATP hCG

> 800

= 400 2

Modulation of ovarian adenylate cyclase response to gonadotropins following luteinization of the rat ovary.

Modulation of Ovarian Adenylate Cyclase Response to Gonadotropins Following Luteinization of the Rat Ovary C. Y. LEE* Department of Laboratory Medicin...
605KB Sizes 0 Downloads 0 Views