BIOLOGY OF REPRODUCTION 50, 765-773 (1994)

Acute Effects of Prostaglandin F2a, Luteinizing Hormone, and Estradiol on Second Messenger Systems and on the Secretion of Oxytocin and Progesterone from Granulosa and Early Luteal Cells of the Ewe1
23 P.A. DENNING-KENDALL ' and D.C. WATHES 4

Department of Anatomy, 3 School of Medical Sciences, Bristol BS8 1TD, United Kingdom Department of Cellular Physiology,4 AFRC Babraham Institute, Cambridge CB2 4AT, United Kingdom ABSTRACT
Previous reports have suggested that gonadotropins, estradiol, and prostaglandin F,, (PGF,,) have varying effects on progesterone and oxytocin synthesis or secretion in cultured granulosa and luteal cells collected at different stages of the estrous cycle. The experiments reported here were designed to investigate whether effects of these agonists on secretion of hormones and their coupling to second messenger systems changed around the time of ovulation. Granulosa cells and Day 2 luteal cells of the ewe were cultured for three days and then treated for 30 min with varying doses of PGF,,, LH, or estradiol. LH increased intracellular cAMP at both stages, but granulosa cells were more responsive in terms of both minimum effective dose (10 compared with 100 ng/ml) and degree of stimulation. LH caused no change in intracellular inositol phosphate levels. Both granulosa and early luteal cells responded to LH treatment by an increase in progesterone output in a dose-responsive fashion. PGF,, increased inositol phosphate accumulation in cells collected at both stages of the cycle. All doses tested (10 - 6 -10 -8 M) stimulated the release of oxytocin into the culture medium from both granulosa and luteal cells. Progesterone secretion was also increased, but only at the highest dose (10 - 6 M). Estradiol treatment (10 - 6 M) did not affect either the inositol phosphate or cAMP second messenger systems, but it did inhibit the secretion of oxytocin from granulosa cells. It is concluded that PGF,, stimulates oxytocin secretion in vitro via activation of the inositol phosphate/Ca2+ second messenger pathway, and that LH increases progesterone synthesis acting via cAMP in both luteinized granulosa and early luteal cells, whereas estradiol does not work through either of these pathways. These data suggest that previously reported changes in the chronic effects of gonadotropins, PGF,,, and estradiol on oxytocin synthesis during the periovulatory period cannot be explained in terms of activation of alternative signalling pathways. The short-term stimulatory effects of PGF,, on progesterone output differ from its reported inhibitory effect in the mature ovine CL and support the idea that granulosa/luteal cell responsiveness to the same agonists alters during luteal development.

INTRODUCTION The preovulatory ovine follicle synthesizes estradiol under gonadotropin stimulation. As the follicle matures, increasing numbers of LH receptors develop on both the thecal and granulosa layers, reaching a maximum during the ascending limb of the LH surge but falling after the peak; FSH receptor concentrations, on the other hand, remain unchanged during this period [1, 2]. At the same time, follicular production of estradiol falls and progesterone increases [1, 3]. Concomitant with this alteration in steroidogenesis is a change in the profile of peptide hormones that are synthesized and secreted, the inhibin gene being downregulated at the same time that the oxytocin gene is switched on [4]. After ovulation the granulosa cells develop into "large" luteal cells (> 22 m in diameter), whereas the thecal cells become "small" luteal cells (10-20 m). Both of these cell types are capable of progesterone production [5, 6]; only the large luteal cells, however, secrete oxytocin [7, 8]. Studies based mainly on CL collected in midcycle have shown that the two luteal cell types differ in their hormonal
Accepted November 10, 1993. Received March 12, 1993. 'Supported by the AFRC. 2 Correspondence: Dr. D.C. Wathes, Department of Animal Health, The Royal Veterinary College, Boltons Park, Hawkshead Rd, Polters Bar, Herts, EN6 1NB, UK. FAX: 0707-647085.

responsiveness. Basal progesterone output is greater from large than from small cells; and although both possess LH receptors, only the small cells respond to LH or cAMP by an increase in progesterone production [9, 10]. The luteolytic prostaglandin F2, (PGF2 ,) causes acute inhibition of progesterone output by large cells of the ewe via activation of protein kinase C [11]. The two second messenger systems may also interact. LH has been shown to increase inositol phosphates and intracellular calcium ([Ca 2 +]i) in bovine luteal cells of the midluteal phase and early pregnancy [12,13], although similar actions of gonadotropins in the ewe are less clear [14]. The relative importance of these different control mechanisms to the overall output of progesterone at different stages of the estrous cycle has not been assessed. The mechanisms involved in oxytocin production and release have been examined in both granulosa and luteal cells. Experiments in vitro have used slices from mature CL, in which short-term exposure to PGF2a would be expected to stimulate oxytocin release via an increase in inositol phosphates and [Ca 2+]i triggering granule exocytosis [11, 15]. However, results have proved equivocal in both the ewe and the cow; some have reported that oxytocin release was stimulated whereas others found no effect, although the tissue did respond to arachidonic acid, phospholipase C, and increases in [Ca2+]i [16-21]. In cultured granulosa cells from preovulatory follicles, oxytocin output is stimulated by di-

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DENNING-KENDALL AND WATHES

butyryl cAMP (dbcAMP) and by activators of cAMP, includ-

ing LH, FSH, and forskolin [22-26]; but shortly before ovulation, the influence of FSH [25,27] and estradiol [28] becomes inhibitory. Continuous exposure of cells to PGF2 ,, also results in a switch from a stimulatory to an inhibitory effect on oxytocin output after ovulation [24]. These granulosa cell studies all involved measuring oxytocin in the medium on a 24-h basis in cells receiving continuous exposure to the various agonists; it was therefore not possible to differentiate effects on oxytocin synthesis and release. The results do, however, suggest that the mechanisms regulating oxytocin output alter around ovulation, and it has been suggested [24] that this could reflect a change in second messenger activation. The aim of the present study was to examine the effects of three different types of agonists, i.e., LH, PGF 2,, and estradiol, on the intracellular production of inositol phosphates and cAMP in both luteinized granulosa cells from preovulatory follicles and Day 2 CL in order to see whether there were any alterations in the coupling to second messenger systems around the time of ovulation. The resultant effects on the secretion of oxytocin and progesterone were also compared at these two stages of the cycle. Cells from each follicle and CL were cultured separately, and all parameters were measured in the same batch of cells.
MATERIALS AND METHODS Animals

transferrin-sodium selenite media supplement [ITS], BSA, DNase, and estradiol) were from Sigma Chemical Co. (Poole, Dorset, UK).
Other Reagents

Myo-2-[ 3H]inositol with 3 [ H]inositol marker set (1

PT6-271 (19.1 Ci/mmol), a ,uCi each of [3H]-IP1 , -IP 2, and ACS aqueous mg), and (100 SAX minicolumns ), Amprep -IP 3 scintillant were obtained from Amersham International (Bucks, UK). Dowex anion-exchange resin AG 1-X8 200400 mesh, formate form was obtained from Bio-Rad Laboratories (Hemel Hempstead, Herts, UK).
Cell Culture

Mature Clun Forest or Dorset Horn ewes were kept at pasture and provided with hay and water ad libitum.-During the breeding season, groups of ewes (n c 16 in total) were fitted with intravaginal Veramix sponges for 12 days (Upjohn Ltd., Crawley, West Sussex, UK; each sponge contained 60 mg medoxyprogesterone acetate). Fourteen days after removal of the sponge (midluteal phase), estrus was synchronized by injection of 100 Eig cloprostenol i.m. (PGF analogue; Coopers Animal Health Ltd., Crewe, Cheshire, UK). Ewes were killed 65 h or 96 h after the prostaglandin injection for collection of preovulatory follicles or Day 2 CL, respectively. The ovaries were removed within 10 mih of slaughter, placed in sterile basic medium (see below), and brought back to the laboratory on ice (approximately 40 min).
Culture Reagents

Dulbecco's Modified Eagle Medium (DMEM)/Ham's F12 mix (with L-glutamine and phenol red) and fetal calf serum (FCS) were supplied by Gibco-BRL (Paisley, Scotland). Collagenase was Worthington Type 1 (Worthington Biochemical Corporation, Freehold, NJ), supplied by Lorne Diagnostics (Reading, Berks, UK). Bovine LH (bLH-B-5) was kindly supplied by NHPP (Baltimore, MD). All other culture reagents (benzylpenicillin, streptomycin sulphate, insulin-

Whole ovaries were rinsed in 70% ethanol and transferred into basic culture medium (DMEM/F-12 containing 5 IU/L penicillin, and 50 mg/L strep1.125 g/L NaHCO 3, 10 tomycin, pH 7.3). Preovulatory follicles or CL were dissected out and placed in fresh medium. Follicular fluid was aspirated, the follicles were cut in half, and the granulosa cells were rinsed out using a sterile plastic pipette. CL were chopped finely and then subjected to one 75-min digestion in basic medium containing 0.2% collagenase, 0.005% DNase, and 0.5% BSA in a shaking water bath at 37C as described previously [29]. In each case the number of viable granulosa or luteal cells was estimated using trypan blue. A total of 11 follicles were obtained from nine ewes; 11 CL were obtained from seven ewes. The cells from each follicle or CL were cultured separately. All available cells were divided equally among the required number of wells, each containing 1.0 ml of medium. This gave a starting number of 5 cells per well. Cells did not reach con4 and 10 between 10 fluence, and the difference in initial plating density did not influence results. Cells were cultured in 24-well multiwell plates (Linbro; Flow Laboratories, High Wycombe, Bucks, UK) in a humidified incubator at 370C in a 5% CO2/air atmosphere. For the first 24 h, basic medium supplemented with 10% FCS was used to promote cell attachment. This was replaced by basic medium containing 1% (v/v) ITS to give final concentrations of 6.25 mg/L insulin and transferrin, 6.25 pLg/ L selenious acid, and 1.25 g/L BSA. After a further 24 h in culture, the medium was replaced with medium containing 5 R.tCi/well myo-2-[ 3H]inositol. The 48-h pretreatment period was chosen to allow for up-regulation of the oxytocin gene in the follicular cultures [4, 28] and to allow for recovery of the plasma membranes of the luteal cells from the collagenase treatment. After a total of 72 h in culture, the cells were incubated according to the method of Brown et al. [30]. First, cells were treated for 45 min at 37C with 0.5 ml sterile PBS, pH 7.4 to remove as much as possible of the unincorporated [3H]inositol; then cells were incubated for 30 min at 37C in PBS containing 10 mM LiCl with or without the test reagents LH, PGF2., or estradiol. The

SECOND MESSENGER SYSTEMS IN THE OVINE OVARY

767

i
e P. U

-O-

INOSITOL PHOSPHATES

(a 2
I-

10 FRACTION NUMBER

20

FIG. 1. Separation of 10500 cpm of myo-2-[ 3 H]inositol (open circles) 3 and 11 600 cpm of a mixture of [3H]IP,, [3H]IP2, and 1 H]IP3 (solid squares) on a 100-mg Amprep SAX minicolumn. After equilibration of the column with 5 ml 1.0 M KHCO 3 followed by 15 ml water, the sample was applied diluted in 4 ml water. [3Hllnositol was eluted with 6 ml water. The column was washed with a further 4 ml water, and inositol phosphates were eluted as a single peak with 4 ml 0.25 M KHCO 3. Fractions of 1 ml were collected.

Measurement of Inositol Phosphates Amprep SAX minicolumns were conditioned with 5 ml 1.0 M KHC0 3 followed by 15 ml water. The extracts (0.49 ml) from each culture well containing inositol phosphates were diluted in 3.51 ml water and applied to the column, at a flow rate of < 5 ml/min, in two 2-ml aliquots followed by 2 ml water. The eluates, which contained free inositol, were collected directly into scintillation vials. The columns were washed with 4 ml of water, which was not collected, followed by two 2-ml aliquots of 0.25 M KHC0 3 to elute inositol mono, bis-, tris-, and tetrakisphosphates (IP 1, IP2, IP3, and IP4) as a single peak. The five 2-ml eluates from each sample were mixed with 8 ml ACS scintillant for 3H counting. Figure 1 shows elution of standards on a SAX minicolumn. Recoveries of inositol and inositol phosphates were > 95%, and the coefficient of variation between duplicate inositol phosphate estimations was 8.4%. Identification of Inositol Phosphates To validate that the eluates obtained as described above contained IP,, IP2, and IP3, two duplicate samples were also applied to the more conventional Dowex resin columns that had previously been characterized by means of [3H]inositol phosphate markers. Inositol phosphates were separated by anion-exchange chromatography by the method of Berridge et al. [35] with minor modifications as described below. Extracts were diluted to 1 ml with PBS and then applied to Bio-Rad polyprep disposable columns (0.8 x 4 cm) containing 1 ml formate-form Dowex AG 1-X8 equilibrated with water. The loaded columns were washed with a) 15 ml water to remove inositol and then b) 5 ml 60 mM sodium formate to elute glycerophosphoinositol. IP 1, IP2, and IP3 were eluted sequentially with 10 ml of c) 0.1 M formic acid:0.2 M ammonium formate, d) 0.1 M formic acid:0.4 M ammonium formate, and e) 0.1 M formic acid:1.0 M ammonium formate, respectively. Column eluates were collected as 1-ml fractions directly into scintillation vials, to which was added 10 ml of ACS scintillant. Samples were then counted for radioactivity. Figure 2 shows the elution profile of two samples together with elution positions of [3 H]-labeled standards. Use of the Dowex columns not only validated the use of the SAX columns, but also showed that the two methods yielded the same result. Samples from one follicle analyzed by the two methods gave measurements of total inositol phosphates in control cells (cpm/105 cells) of 578 and 645 for Dowex and SAX columns, respectively; these measurements increased to 3653 and 3564, respectively, after PGF 2. treatment. The Dowex columns also showed that the granulosa cells contained IPI > IP2 > IP3, with IP2 and IP3 barely detectable under resting conditions. After stimulation with PGF2 , the amount of radioactivity in all three IP fractions increased (Fig. 2). Measurement of cAMP Extracts (0.05 ml) from each culture well were diluted with 0.3 ml 0.05 mM sodium acetate, pH 5.8. Diluted sam-

LiCl was added to inhibit inositol phosphatase and thus to allow for the accumulation of IP as well as IP2 and IP3 [31]. In the first series of experiments, LH (100 ng/ml; 3.1 x 10 - 9 M), PGF2Q (476 ng/ml; 1 x 10-6 M), and estradiol (200 ng/ml; 0.74 x 10 - 6 M) concentrations were chosen to represent physiological levels measured during the LH surge [32] and in follicular fluid of preovulatory follicles [2, 33]. In a second series of experiments, a range of LH (100-1 ng/ml) and PGF2,, (10-610 - 8 M) doses were tested. After hormone treatment the solutions were removed by suction and replaced by 0.5 ml ice-cold 5% (v/v) perchloric acid, and the culture plates were kept at 4C for 30 min to extract cellular cAMP and inositol phosphates [34]. The complete contents of each well were transferred to a polypropylene tube containing 0.12 ml of 10 mM EDTA (pH 7.0). The samples were neutralized by the addition of 0.55 ml of a 1:1 (v/v) mixture of 1,1,2,-trichloro-trifluoroethane and tri-noctylamine (Sigma). After centrifugation for 2 min at 800 x g, the upper phase (0.54 ml), which contained cAMP (recovery 85%) and inositol phosphates (recovery 88%), was stored at -20C in two separate polypropylene tubes containing 0.49 ml and 0.05 ml for subsequent analysis of inositol phosphates and cAMP, respectively. All media and PBS from treated cells were stored at -20C for subsequent analysis of oxytocin and progesterone by RIA. Each culture also had one extra well for the determination of DNA. This well did not receive any [3H]inositol or test reagents, but after 72 h in culture the medium was removed and the cells were washed twice with cold PBS and extracted for 15 min at 370C with 0.5 ml 0.2% (w/v) Triton X-100 in 10 mM NaOH. The DNA extracts were stored in silica glass tubes at -20C. The medium from these wells was used for progesterone and oxytocin RIA to assess hormone output before the start of the experimental treatments.

768
I a

DENNING-KENDALL AND WATHES

i I .

I ! l-

3000
Ii
0

2000

U 4 Io

IOL 1000

0 0 10 20 30 40 50 60 FRACTION NUMBER
FIG. 2. Elution profiles of extracts of granulosa cells from a preovulatory follicle. Controls (open circles) received PBS only; PGF 2,-treated cells (solid squares) received PBS containing 1 x 10-6 M PGF 2. for 30 min. Extracts were applied in 1 ml PBS to a Dowex AG 1-X8 column (containing 1 ml resin) equilibrated with water. The loaded columns were washed with: a) 15 ml water, b) 5 ml 60 mM sodium formate, c) 10 ml 0.1 M formic acid:0.2 M ammonium formate, d) 10 ml 0.1 M formic acid:0.4 M ammonium formate, and e) 10 ml 0.1 M formic acid:1.0 M ammonium formate. Column eluates were collected as 1-ml fractions directly into 10 ml ACS scintillant before counting. Arrows show elution positions of 3H]IP1, IP 2, and IP3 standards.

ples were assayed in a [1251]-cAMP scintillation proximity assay using rabbit antisuccinyl cAMP serum (Amersham International). The sensitivity of the assay was 2 fmol (0.7 pg)/ tube. The intraassay coefficient of variation was 9%. RlAs Oxytocin and progesterone were measured in unextracted samples of culture medium as described previously [18]. Crossreactivities of the antisera used were as follows: 1) oxytocin-mesotocin (1.0%), arginine vasotocin, arginine vasopressin, and neurophysin I and II, all < 0.001%; 2) progesterone--ll -hydroxypreg-4-ene-3,20 dione (74%), 11 -hydroxypregn-4-ene 3,20 dione (10%), testosterone

(0.06%), androstenedione (0.03%), estradiol-170f, estrone, and pregnenolone, all < 0.01%. The intra- and interassay coefficients of variation were 8.2% and 13%, respectively, for oxytocin and 2.1% and 8.7%, respectively, for progesterone. Determnnination of DNA The cell number after culture was estimated by measurement of DNA using calf thymus DNA standard and assuming 1.67 x 105 cells to contain 1 ,ug DNA. Through use of the method of Sorger and Germinario [36], cells were solubilized in an alkaline Triton X-100 solution without sonication or homogenization. Standard DNA or solubi-

a TABLE 1. Hormone secretion by preovulatory granulosa cells and Day 2 luteal cells during a 3-day culture period.

% Increase

Oxytocin secretionb

No. of follicles
or CL

in cell number during

cultureb Day 1

fmol/104 cells/24 h
Day 3c Day
3d

Progesterone secretionb

pmol/10 4 cells/24 h
Day 3
c

Day 1

Day 3d

Granulosa Luteal

7 7

427 88 143 + 24

56 - 11 157 -+36

114 + 23* 66 + 13*

323 + 61** 83 + 13*

111 35 204 + 16

182 37* 17 3**

615 - 200* 29 - 10**

'Cells were plated in 10% FCS. This was replaced by defined medium on Day 1. Medium was changed daily. bCell numbers on Day 1 were the number of viable cells plated as assessed by trypan blue; cell numbers on Day 3 were calculated from DNA measurements. cdHormone release into the medium has been calculated on the basis of the number of cells actually present on Day 3 (superscript c) and also on the number of cells originally plated (superscript d). All values are the mean + SEM. Significant differences in hormone secretion rates between Days 1 and 3 are shown as follows: *p < 0.05, **p < 0.01.

SECOND MESSENGER SYSTEMS IN THE OVINE OVARY

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TABLE 2. Effect of short-term (30 min) treatment with LH, PGF 2 , or estradiol on the intracellular concentrations of cAMP and inositol phosphates and on the secretion of progesterone and oxytocin by ovine granulosa cells. Treatment Second messenger/hormone secretion in control culturesa LH 100 ng/ml (%of control)b PGF 2. 10-6 M (%of control) b Estradiol 200 ng/ml (%of control)b cAMP (fmol/10 5 cells) 100 (64-130) 1126%* Total IPs (cpm/10 5 cells) 774 (112-930) 105% + 6 826%0/*** +119 91% +16 Progesterone (pmol/10 5 cells) 33 (19-51) 131%*** Oxytocin (fmol/10 5 cells) 239 (7-622) 114% + 6 164/* +16 70%* +5

443
118% 19 110% + 5

6
134%*** 5 100% +8

aResults are given as the mean of seven separate preovulatory follicles with the range in parentheses. bResults show the mean + SEM %values following a 30-min treatment expressed as %of control where control = 100%. Significance values were calculated on raw data using 2-way ANOVA: *p < 0.05; **p < 0.025; ***p < 0.01.

lized material was then added directly to the fluorescent dye diamidinophenylindole (DAPI). Minor modifications were made: cells were solubilized with 0.5 ml Triton solution; the standards ranged from 0.0625 to 2.5 ,ug DNA/ ml; and half the recommended concentration of DAPI was used in order to increase assay sensitivity. Standards were assayed in triplicate and samples in duplicate. StatisticalAnalyses Hormone secretion rates between Days 1 and 3 in culture were compared by paired t-test. Comparisons between control and treated cells in terms of secretion of oxytocin and progesterone and intracellular concentrations of cAMP and inositol phosphates over the 30-min test period were carried out using two-way analysis of variance of log-transformed data. The linear relationship of hormone output with log dose of LH and PGF2,, was also determined.
RESULTS

In the present study we have compared endocrine responses of cultured granulosa and early luteal cells to examine potential changes in control mechanisms associated with early luteal development. The granulosa cells were collected from individual preovulatory follicles 65 h after a luteolytic dose of cloprostenol. This time point occurs after the start of the LH surge (which begins 48-66 h after PGF2a in our flock) and shortly before ovulation. The cells luteinized in vitro as demonstrated by their ability to produce increasing amounts of progesterone over the 3-day culture period (Table 1). The early luteal cells were obtained on Day 2 (approximately 24 h after ovulation) and contained a mixed population of granulosa- and theca-derived cells, as these cannot be separated by size at this stage of the cycle. The characteristics of unstimulated granulosa and luteal cells were different in culture. There was at least a doubling of the granulosa cell number over 72 h in six out of

seven cultures and, in two cases, a 7-fold increase. There was a concomitant doubling in oxytocin secretion per cell over the 3-day culture period (Table 1) with a similar rise in progesterone. In contrast, only two of seven luteal cell cultures showed any increase in cell number (each approximately 2-fold over this period); and all these cultures secreted the most oxytocin and progesterone on the first day of culture, with oxytocin output per cell halving between Days 1 and 3 and progesterone secretion showing a 10-fold decrease. For measurements of inositol phosphates the SAX minicolumns proved very efficient, although IP 3 was not fully resolved from the fraction containing 1P, and IP 2 when used according to the manufacturer's instructions. The columns were therefore used to measure the [3 H]inositol content and the total [3 H]inositol phosphates. In control granulosa and luteal cells, most (> 98%) of the radioactivity was found as free [3 H]inositol, with inositol phosphates containing 1.5% and 1.2% of the radioactivity, respectively. Resting concentrations of both cAMP and inositol phosphates were similar between the cultured granulosa and luteal cells (Table 2); they were also within the same range as previously reported values for cultured bovine luteal cells [12]. Table 2 shows the effect of short-term (30 min) treatment with maximal doses of LH, PGF2., and estradiol on both second messenger systems and hormone release in luteinized granulosa cells from preovulatory follicles; Figure 3 depicts dose-response effects from the second series of experiments. LH at 100 ng/ml stimulated a large but variable (2-34-fold) increase in cAMP but had no effect on inositol phosphates. A dose of 10 ng/ml stimulated all three follicles tested, whereas 1 ng/ml was stimulatory in only one of three follicles. The rise in cAMP was associated with a significant dose-responsive increase in progesterone secretion. In the first series of experiments, LH did not affect oxytocin release; but in the second series, the 100 ng/ml dose caused a small increase in secretion. PGF2. had no effect on cAMP but stimulated inositol phosphate accumu-

770
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1 ng/ml
4a
Io C

10 ng/ml 100 ng/ml

3C 2C 1a

o
0

cAMP

IP

PROG

OXT

cAMP was still significant, although the increase was much smaller and LH was effective only at the highest dose. LH nevertheless had similar effects on hormone secretion, causing an increase in progesterone output with no effect on oxytocin. PGF2a stimulated a major increase in inositol phosphate accumulation at all three doses. In the first series of experiments, the highest dose, 10- 6 M, caused a minor but highly significant rise in cAMP; there was a similar trend in the second series employing a range of doses, but this did not achieve significance, possibly due to the smaller number of CL analyzed. The changes in inositol phosphate were associated with increased secretion of both progesterone and oxytocin. As for granulosa cells, this occurred at a lower PGF2 , dose for oxytocin (10 - 8 M) than for progesterone (10-6 M). Estradiol caused no short-term alteration in either second messenger system or hormone release. DISCUSSION

Corpora

lutea

So -

o o 0 0
CO

cAMP

IP

PROG

OXT

FIG. 3. Effects of a range of doses of LH on second messenger and hormone release by luteinized granulosa and early luteal cells in culture. Results show the mean + SEM values for cells from three or four individual follicles or CL after a 30-min treatment, expressed in relation to control values of 100%. Significant increases from control values were calculated on log-transformed raw data using two-way ANOVA. Significance values on the bar above each data set indicate linear dose-response effects. *p < 0.05, **p < 0.01, ***p < 0.001.

lation in a dose-responsive fashion. This was associated with the increased release of both progesterone and oxytocin -6 into the medium. However, all three doses (10-8-10 M) -6 stimulated oxytocin release, whereas only the highest (10 M) dose increased progesterone output. Estradiol at 200 ng/ml affected neither cAMP, inositol phosphates, nor progesterone but caused a significant inhibition of oxytocin release (Table 2). Results from the early luteal cells are presented in Table 3 and Figure 4. In the early luteal cells the effect of LH on

The times of collection of the granulosa cells and luteal cells used in the present series of experiments were only 30 h apart in relation to the cloprostenol injection, but the cells showed several distinct differences in their behavior in vitro. Granulosa cells continued to proliferate actively whereas luteal cells did not usually increase in cell number over three days. Granulosa cells were able to increase their output of both progesterone and oxytocin with time in vitro, whereas the secretion of both hormones from the luteal cells declined between Days 1 and 3. These data are consistent with previously reported findings [4, 24, 25] and confirm that the ability of the cells to up-regulate their oxytocin synthesis is acquired before ovulation [28]. The increase in oxytocin output with time probably represents synthesis, as both mRNA and hormone concentrations are virtually undetectable in uncultured cells [4, 28]. The divergent pattern of progesterone production over time between granulosa and luteal cells may reflect changes in steroid metabolism after ovulation. Granulosa cells are unable to utilize lipoprotein-derived cholesterol as a steroid precursor, possibly due to a lack of high density lipoprotein (HDL) binding sites, and instead rely on de novo synthesis of cholesterol [37]. After ovulation, the luteal cells become dependent on vascular-derived lipoproteins to supply their cholesterol requirements [38, 39]. The fall in basal progesterone output of the luteal cells between Days 1 and 3 may therefore reflect the withdrawal of FCS as a cholesterol source after the first 24 h in vitro. The experiments reported here were designed to test the effect of a short-term exposure to various agonists on endocrine responsiveness during the periovulatory period. Previous workers have generally measured hormone release from cultured bovine and ovine granulosa and luteal cells over a 24-h period [4,18, 24] or alternatively have examined output from luteal slices during 1-3-h incubations

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TABLE 3. Effect of short-term (30 min) treatment with LH, PGF 2., or estradiol on the intracellular concentrations of cAMP and inositol phosphates and on the secretion of progesterone and oxytocin by ovine luteal cells. Treatment Second messenger/hormone secretion in control cultures' LH 100 ng/ml (%of control)b PGF 2 10 6 M (%of control)b Estradiol 200 ng/ml (% of control)b
-

cAMP (fmol/105 cells) 153 (104-219) 129%**

+9

Total IPs (cpm/10 5 cells) 448 (124-645) 97%

+ 6

Progesterone (pmol/105 cells) 25 (8-56) 124%/o*** +5 177%*** 17 91%

+5

Oxytocin (fmol/105 cells) 158 (75-360) 115%

+6

137%** 9 112%
+12

900%** +177 92%

+ 7

360%* -98 104% 11

aResults are given as the mean of seven separate Day 2 CL with the range in parentheses. bResults show the mean - SEM %values following a 30-min treatment expressed as %of control where control = 100%. Significance values were calculated on raw data using 2-way ANOVA: *p < 0.05; **p < 0.025; ***p < 0.01.

[16,17, 40]. The present system has two advantages for the study of oxytocin. Firstly, the use of a short (30 min) time period allows differentiation between acute effects on hormone release and longer-term actions on hormone synthesis. Secondly, the use of cultured granulosa and early luteal cells that are actively synthesizing oxytocin avoids the problem of luteal cell degranulation that occurs during dispersion of mature luteal cells [18, 41]. Treatment with LH increased cAMP concentrations in both granulosa and luteal cells, but the effect was about 10-fold greater, although variable, in the granulosa cells. In the ewe, granulosa cell LH receptor concentrations increase during the preovulatory period, peak during the LH surge, and then decrease abruptly [2]. In the rat, hCG down-regulation of ovarian LH receptors is maximal 12-24 h after treatment [42]. Therefore, differences in responsiveness between individual follicles and between granulosa and early luteal cells are likely to reflect changes in LH receptor binding with time after the LH surge. Despite the quantitatively smaller cAMP increase in the luteal cells, both granulosa and luteal cells showed a similar rise in progesterone output. Granulosa-derived large luteal cells from mature CL are now known to possess LH receptors; but they are not thought to respond to LH by increasing progesterone production [11], possibly due to an unavailability of appropriate phosphoprotein substrates for A kinase [43]. Therefore the pronounced response to LH in terms of an increased progesterone output seen in our early luteal cell cultures can probably be attributed to the thecal/small luteal cell contribution. Its physiological relevance at this stage of the cycle may not be great, however, as the effect occurred only at doses of 10-100 ng/ml, whereas peak circulating LH concentrations at this time rarely exceed 3 ng/ml. We found no effect of LH on inositol phosphates in either granulosa or luteal cell cultures. This is in contrast to the work of Davis et al. [12, 13], who demonstrated in isolated small luteal cells from mature bovine CL a rapid and sustained rise in inositol phosphates in response to LH. It is not clear

whether this difference is due to species, age of CL, or length of time between cell isolation and treatment. Previous studies by various groups have demonstrated that long-term treatment of bovine and ovine granulosa cells with LH, FSH, or dbcAMP usually stimulates oxytocin output except in the immediate periovulatory period, when the effect of FSH is reversed [22, 23, 25, 27]. These responses were measured over 24-h periods, and they could not consistently be repeated after an LH-induced increase in cAMP in the present experiments using a 30-min exposure time. This suggests that cAMP is acting on the biosynthetic pathway of oxytocin synthesis rather than on the acute release of pre-formed granules. Recent studies on ovarian oxytocin gene expression indicate that it is inhibited by binding of a COUP transcription factor to the gene promoter region during most of the cycle but that this is displaced by another transcription factor during the periovulatory period, leading to up-regulation [44]. This competing factor has yet to be identified but is likely to be responsive to cAMP. Treatment with PGF2. caused a large increase in the inositol phosphate content of both granulosa and luteal cells, and the release of both progesterone and oxytocin into the medium was increased in both cell types. Although PGF2, clearly stimulates the release of luteal oxytocin when administered in vivo [45], results of previous in vitro studies have proved inconsistent (see Introduction). It is possible that previous efforts to show this effect in luteal slices in short-term incubations failed because exposure to high concentrations of endogenously released PGF 2a during tissue collection causes an initial down-regulation of the luteal PGF2, receptor [46]. In previous work, chronic exposure of bovine granulosa and early luteal cells to PGF2a produced opposing results in that granulosa cell output of oxytocin over 24-h time periods was increased whereas that of luteal cells was inhibited [24]. On the basis of these data, McArdle [24] suggested that PGF2, might be acting via cAMP-coupled PGE receptors in the granulosa cells in contrast to protein kinase C-cou-

772

DENNING-KENDALL AND WATHES

300
-a

3F 108 M 3F 10M GF 1M

40
U

200

100

cAMP

IP

PROG

OXT

---zuuu Corpora 1500

0
(a
'U

lutea

**

10

1000

***
Tr
* *-

500

0 cAMP IP PROG OXT

FIG. 4. Effects of a range of doses of PGF 2, on second messenger and hormone release by luteinized granulosa and early luteal cells in culture. Results show the mean SEM values for cells from three or four individual follicles or CL after a 30-min treatment, expressed in relation to control values of 100%. Significant increases from control values were calculated on log-transformed raw data using two-way ANOVA. Significance values on the bar above each data set indicate linear dose-response effects. *p < 0.05, **p < 0.01, ***p < 0.001.

pled PGF2,, receptors in luteal cells. We have now demonstrated that 10-8 M PGF2, increases inositol phosphates, but not cAMP, in luteinized granulosa cells, showing that this explanation is probably incorrect. The difference between the inhibitory effect of PGF2, on oxytocin output by cultured bovine Day 2 luteal cells [24] and the stimulation shown here is again likely to reflect differences between synthesis (measured over a 24-h period) and acute release. Previous studies of the action of PGF2a on the short-term progesterone response of luteinized granulosa and luteal cells have been inconsistent, reporting a stimulation in lu-

teinized bovine granulosa cells [47], a stimulation in small but not large bovine luteal cells [13, 48], and no effect on small, but an inhibition in large, ovine luteal cells [11]. With our protocol we observed stimulation in both granulosa and early luteal cells, although the latter were more responsive. The reasons for these variations between groups are obscure but may in part reflect an age-associated change from stimulatory effects in young CL to inhibitory actions in more mature ones. A higher PGF2a dose was needed to influence progesterone than to influence oxytocin output in both cell types. This suggests the possibility that low concentrations of PGFz2 produced endogenously by the CL could regulate oxytocin release during the early luteal and midluteal phases without affecting steroidogenesis. Short-term estradiol treatment had no effect on either cAMP or inositol phosphates in either granulosa or luteal cells, although in the uterus, estrogens may stimulate the production of inositol-containing phospholipids [49] and recent studies have shown the rapid release of [Ca 2+]i in cultured chicken and porcine (but not rat) granulosa cells in response to estradiol-173 stimulation, an effect probably mediated by phosphoinositide breakdown [50]. In the present experiment it is unlikely that [Ca2+]i was increased by estradiol, as treatment decreased rather than increased oxytocin release from the granulosa cell cultures. We have previously demonstrated that chronic exposure of granulosa cells to estradiol-173 can either increase or decrease oxytocin output depending on the time of follicle collection in relation to ovulation [28]. In these previous experiments the medium was changed at 24-h intervals, the effects were noticeable only after 3-5 days in vitro, and the effects involved marked alterations in oxytocin mRNA expression ([28] and Wathes and Denning-Kendall, unpublished observations). The mechanism of action is therefore likely to differ from that seen with a 30-min exposure period, in which nongenomic actions on the plasma membrane are more probable. In conclusion, we show here that LH and PGF2 ,, remained coupled to the cAMP and inositol phosphate second messenger systems, respectively, in both luteinized granulosa and early luteal cells. In addition, LH had consistent short-term stimulatory effects on progesterone synthesis by ovine granulosa and early luteal cells, and the acute administration of PGF2a caused both oxytocin and progesterone release. ACKNOWLEDGMENTS
We thank the National Hormone and Pituitary Program, Baltimore, MD, for the gift of bLH; Dr. A.N. Corps for helpful comments on the manuscript; Mr. G. Davies for care of animals, and Mrs. J. Skinner and Mrs. J. Brown for preparation of the manuscript.

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