Molecular and Cellular Endocrinology, 84 (1992) 137-143 0 1992 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/92/$05.00
137
MOLCEL 02716
Prostaglandin F,, and gonadotropin-releasing hormone increase intracellular free calcium in rat granulosa cells Marie R. Rodway ‘, Gillian L. Steele a, Kenneth G. Bairnbridge b and Peter C.K. Leung ’ aDepartments of Obstetrics and Gynaecology and ’ Physiology, Uniuersity of British Columbia, Vancoucer, Canada (Received 12 September 1991; accepted 2 December 1991)
Key words: Prostaglandin F,,; Gonadotropin-releasing
hormone; Granulosa ceil; Calcium
Summary Changes in cytosolic free calcium concentration ([Ca2+]i) in response to prostaglandin F,, (PGF,,) and gonadotropin-releasing hormone (GnRH) were measured in single rat granulosa cells, using the calcium-sensitive fluorescent dye, fura-2AM. In 90 out of 135 granulosa cells (67%), there was a 3- to 4-fold increase in resting [Ca’+]i within 30 s of administration of PGF,, (10e6 M). The resting [Ca2+li returned to pre-stimulation levels in approximately 80 s. Granulosa cells were responsive to PGF,, at concentrations ranging from 10V7 M to lop4 M (n = 7). Within this range of concentrations, the magnitude of the calcium response did not differ. In another series of e~eriments, the majority (93%, n = 57) of the granuiosa cells which responded to PGF,, also responded to GnRH. Neither the magnitude of the [Ca2+li response nor the time to response differed between PGF,, and GnRH. Furthermore, simultaneous treatment of granulosa cells with both hormones did not generate a larger response than with either hormone alone. During perifusion with low calcium media, the characteristic [Ca”]i response to PGF,, decreased, and was eliminated within 10 min (n = 91. Similar observations were made in response to GnRH under these conditions. These data confirm that PGF,, and GnRH stimulate a transient increase in [Ca2+li in rat granulosa cells, the source of which may be shared intracellular stores.
Prostaglandin F,, (PGF,,) affects granulosa cell function and is believed to be involved in the
Correspondence to: Dr. Peter C.K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, Grace Hospital, 4490 Oak Street, Vancouver, B.C. V6H 3V5, Canada. This work was supported by grants from the Medical Research Council of Canada to K.G.B. and P.C.K.L.
mechanisms regulating ovulation (A~strong, 1981; Hsueh et al., 1984). Both the preovulatory Iuteinizing hormone (LH) surge and exogenous CAMP stimulate ovarian PG synthesis (Bauminger and Lindner, 1975; LeMaire et al., 1979) in follicles destined to ovulate (Hamberger et al., 1986). Indomethacin blocks this increase in PG synthesis at doses which prevent ovulation (Zor et al., 1977). The effect of indomethacin on ovulation can be reversed by injection of PGs, confirming the importance of PG production at this time (Hamberger et al., 1986; Priddy and Killick, 1988).
138
Furthermore, antisera to PGE, or PGF,, block LH-induced ovulation when administered systemically in mice (Lau et al., 1974), or intrafollicularly in estrous rabbits (Armstrong et al., 1974). In the latter study, antiserum to PGFzII was more effective than that of PGE, (Armstrong et al., 1974). In the rat, GnRH also exerts direct effects on ovarian function (Hsueh and Erickson, 1979; Hsueh and Jones, 1981; Labrie et al., 1981; Clark, 1982; Hillensjo et al., 1982; Knecht et al., 1985; Leung, 1985). Signal transduction of the growth hormone-releasing hormone (GnRH) message in the ovary, as in the pituitary (Chang et al., 1986; Conn et al., 1987), is believed to be via breakdown of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P,) (Naor and Yavin, 1982; Davis et al., 1983; Leung et al., 1983; Ma and Leung, 1985; Minegishi and Leung, 1985). The products of PtdIns(4,5)P, breakdown, diacylglycerol (DAG) and inositol 1,4,5_trisphosphate (Ins(1,4,5)P,), are second messengers which activate protein kinase C (PKC) and increase cytosolic free calcium ion concentrations ([Ca’+&), respectively (Nishizuka, 1988; Berridge and Irvine, 1989). We have previously demonstrated this elevation of [Ca2+li in response to GnRH in rat granulosa cells (Wang et al., 1989). Like GnRH, PGF,, enhances the turnover of phosphatidylinositol in rat granulosa cells (Minegishi and Leung, 19851, suggesting a similar transduction mechanism involving the metabolism of inositol phospholipids and calcium signalling. The purpose of these experiments was to investigate and compare the effects of PGF,, and GnRH on [Ca2+li in individual rat granulosa cells.
Materials
and methods
Animals
Immature Sprague-Dawley female rats (23 days old) were purchased from Charles River Canada (Montreal, Canada) and were injected S.C.with 12 IU pregnant mare’s serum gonadotropin. After 48 h, the rats were killed by cervical dislocation and granulosa ceils harvested as previously described (Minegishi and Leung, 1985).
Cell preparation
For intracellular calcium measurements, granulosa cells (0.1 X 105/ml) were plated in a monolayer onto 18 mm diameter uncoated glass coverslips and incubated in minimum essential medium (MEM; Gibco, Grand Island, NY, USA) containing 5% fetal bovine serum. After 48 h incubation at 37°C in an atmosphere of 5% CO, in air, cells were loaded with 5 PM fura-2-acetoxy-methyl ester (fura-2AM) (Molecular Probes, Eugene, OR, USA), in 1% dimethyl sulfoxide as previously described (Grynkiewicz et al., 1985; Wang et al., 1989). The coverslips were rinsed after 1 h incubation to wash out excess fura-2AM. Fluorescence measurement
Individual coverslips were mounted in a laminar flow-through chamber (volume approx. 350 ~1). Silicone rubber was used to complete a water-tight seal and the chamber inserted into a stainless steel holder. The assembly was then mounted onto the stage of a Zeiss Jenalumar microscope equipped for epifluorescence. The light source was a 200 Watt mercury arc lamp powered by a DC power supply. The light was first passed through one of three differential interference filters (350, 365, or 380 nm, bandwidths of 10 nm) mounted in a turret which could be rotated by a computer-controlled stepping motor. The light was then passed through a 410 nm dichroic mirror and a 100 x apochromat oil immersion lens with a numerical aperture of 1.4 and an adjustable diaphragm to reduce the light intensity. A field diaphragm in the light path prior to the dichroic mirror was used to reduce the area of illumination to the size of a single granulosa cell. All fluorescent light passed back through the dichroic mirror and a 450 nm band pass filter to reduce background fluorescence. The emitted fluorescence was deflected either to the eyepiece or to a camera port in which was mounted a photomultiplier tube, used to convert the fluorescence into a DC voltage. The voltage was converted to digital form and each ratio measurement was taken with an interval of 0.35 s and repeated either at 1.8 or 5 s intervals during hormone treatment. Between hormone treatments, resting [Ca2+li was determined approximately every 1 min to minimize exposure of the
cell to ultraviolet light. The method of measurement with our photomet~ based system would have precluded any observation of high frequency [Ca’+], oscihations, but slower oscillations (> 2 s) would have been observed. The responsiveness of the fura-2AM to changes in [Ca2+Ii was confirmed by direct injections into the laminar flow chamber of 500 PM Br-A23187, a non-fluorescent calcium ionophore (HSC Research Development Corp., Toronto, Canada). The cells could be used for up to 4-5 h after loading with only minimal signs of leakage of fura-2AM. Perifusion with Earle’s balanced salt solution (EBSS), or EBSS with no added calcium (designated low calcium EBSS) was at a rate of 4 mf/min. PGF,, and GnRH were administered directly into the chamber in 500 ~1 aliquots at a concentration of lop6 M. Hormones solubilized in EBSS were washed through with the perifusion medium except where otherwise indicated. Both PGF,, and GnRH were purchased from Sigma (St. Louis, MO, USA). Numbers are reported as mean rtr standard error of the mean. Student’s t-test was used to determine the significance of the difference between resting [Ca2+li in experiments where cells were perifused with low calcium EBSS or EBSS containing PGF,,. Results Changes in [Ca2+ji
4oo r-
-----
t
o
t
‘L__..L_--.._
1
-
.__!__J
0
20 l?ne
30
(min)
Fig. 1. The effect of PGF,, on [Ca2+li in a single rat granulosa ceil. Cells were prepared as described in Materials and methods. PGF,, was administered in a concentration of lo-’ M in 500 &I EBSS at times indicated by arrows in the figure. Similar results were seen in 90 individual cells in 25 experiments (n = 135).
Perifusion of granulosa cells with PGF,, (lo-’ MI in four separate experiments resulted in transient increases in [Ca*+l, similar to those seen with PGF,, administered in 500 ~1 aliquots. As shown in Fig. 3, the time from the start of perifusion until the cells responded was 1.5-2.3 min. The longer delay in response as compared to administration of 500 ~1 aliquots was likely due to the time required to fill the tubing and the chamber with PGF,,. The magnitude of the response was 4.6- to 7-fold, which was similar to the fold response to 500 ~1 aliquots of PGF,, (low6 M) administered in these cells. After 0.5-2.3 min, the [Ca’+]i fell to levels much lower than the
in response to PGF,,
A total of 135 granulosa cells were studied in 25 experiments. 67% of the cells responded to PGF;, (10e6 M) with an increase in [Ca*+J,. The average resting calcium level was 110 + 3.2 nM, the average response was 4.0 + 0.3-fold, the average time to response was 29 + 1.3 s, and to recovery was 82 + 7 s. A representative experiment is shown in Fig. 1. This response was not observed in cells treated with PGE, at low5 M (n = 16) or 1O-4 M (n = 18). The time between administrations of PGF?, was reduced in order to determine the minimum amount of time required to produce responses of equal magnitude. In four trials, the magnitude of the response began to diminish between 3 and 5 min (Fig. 2).
I
600
-10
0
10
20
--I 30
40
Time (mid
Fig. 2. The effect of a decrease in the time interval between administrations of PGF,, on alterations in ]Ca2+li in a single rat granuiosa cell. PGF,, (10m6 M in 500 ~1) was administered at times indicated by arrows in the figure. Similar results were seen in four cells.
140
PCS&
500
perifusion
’
800 GnRH
400 i
c ;;= N
600
_
400
_
h
200
-
2
2
;;+ 1
300
! -
*
200 I
0 100
.. 0,
Time (mid
,
X”
/ 0
Fig. 3. The effect of PGF,,
perifusion on [Ca2’li in a single rat granulosa cell. Cells were perifused with lo-’ M PGF,, (in EBSS) during the time period indicated by the black bar. The time delay to response of the cell was likely due to the time required to fill the tubing and chamber with media. Similar results were seen in four cells.
300
1
~------
t 0
to._
t
--_‘_tL$
t -
t
~-.-
20
-.
30
Fig. 4. An investigation of dose-response relationship of PGF,, in a single rat granulosa cell. PGF,, was administered in various concentrations at times indicated by the arrows in the figure. Seven individual cells produced similar results.
~
-_ ._-.-
Time (mid
Fig. 6. The effect of concurrent administration of GnRH and PGF,, on [Ca”’ Ii in a single rat granulosa cell. PGF,, and GnRH (IO-’ M in 500 ~1) were administered together, then separateiy to the same cell. Similar results were seen in five individual granulosa cells.
peak response, but significantly higher (p < 0.01) than the pre-perifusion resting [Ca2+],.
Time (mid
400
PGs*
i :
~_ll___--------
Investigation of concentration-response relationship and minimum effective concentration Granulosa cells responded to PGF,, with the characteristic increase in [Ca2+ji at concentrations ranging from lo-’ M to lop4 M. The magnitude of response in seven granulosa cells studied did not change when the concentration of PGF,, was increased (Fig. 4). Response to GnRH in cells responsive to PGF,, In another study of 84 granulosa cells, 53 cells (63%) responded to both GnRH and PGF?,; 22
-----
GnRH PGFu
300
no added
Ca2’
f 5 7 200 (u 3
100
0
.A”__L._I__
t 0
t, I
10
--
Time (mid
Fig. 5. The response of a single rat granulosa cell to GnRH and PGF,,. GnRH and PGF,, were administered at a concentration of lo-’ M in 500 JLI EBSS at times indicated by arrows in the figure. The number of cells responding to GnRH previously shown to be responsive to PGF,, was 53 out of 57 (93%). Note that the ICazcli response to PGF,, in this ceil shows small fluctuations.
0
10
Time (mid
Fig. 7. The effect of low calcium perifusion on the response of a single granulosa ceil to PGF,,. PGF,, was administered in a concentration of IO-’ M in 500 1.11EBSS at times indicated by arrows in the figure. Low calcium EBSS was perifused during the time indicated by the black bar. Nine cells responded in a similar fashion.
141
‘**~-1 5 S
h
i
no added Ca*’
t
t
0 0
I i0
6
Time (mini Fig. 8. The effect of low calcium perifusion on the response of a single granulosa cell to GnRH (n = 5). Cells were treated with the agonist (10e6 M in 500 ~1) at times indicated by the arrows. L,ow calcium medium was perifused during the time indicated by the black bar.
cells (26%) responded to GnRH only and four cells (5%) responded to PGF,, only; five (6%) did not respond to either agent. The average fold increase in [Ca2+li in response to GnRH was 3.0 I: 0.4, and the average time to response was 32.8 of:3.1 s (Fig. 5). ~~rn~i~e~ administration of PGF,,
and GnRH
In four experiments, granulosa celIs that responded to both PGF,, and GnRH (lo-” Ml showed no additive increase in [Ca*+], when both agents were administered in the same 500 pl aliquot of EBSS (Fig. 6, n = 9). Lonl calcium perifusion experiments Perifusion of granulosa cells with low calcium EBSS (n = 9) eliminated the [Ca2”li response to PGFZa in appro~mately 10 min (Fig. ‘7,p < 0.01). There was a gradual decrease in this [Ca2+li response over the perifusion period ~unpub~ished observations). The responsiveness of granulosa cells to GnRH was lost in a similar fashion over the same approximate time period following perifusion with low calcium media (Fig. 8, y1= 5). Discussion Using furamicrospectrofluorimetry, this study demonstrates an elevation of [Ca2+], in response to PGF,, in single rat granulosa cells. The response is similar to that reported for GnRH
in the same cell type; a rapid and transient one (Wang et al., 1989). The magnitude of the [Ca2’]i increase, latency and duration of response, as well as the nature of response were indisti~guishable between hormone treatments. There was no effect of increased PGF,, concentration on the magnitude of the response, as observed with GnRH. Furthermore, constant perifusion of the cells with either hormone resulted in a similar single and transient response (Wang et al., 1989). Interestingly, the majority (93%) of rat granulosa cells that responded to PGF,, with an increase in [Ca2+ji also responded to GnRH. Similar to the effects of GnRH on granulosa cells (Wang et al., 1989) and of PGF,, on luteal cells (Rodway et al., 1991) an interesting feature of the effect of PGF,, was that individual granulosa cells failed to demonstrate a dose response. Rather, these cells exhibited an all or none response to treatment with the prostaglandin. This would suggest that a dose-response effect seen when measurements are taken from a large population of cells reflects the recruitment of individual cells to the responding population, rather than each cell contributing more and more to the average [Ca”‘& signal. Further, we observed very littIe evidence that PGF,, consistently induced oscihations in [Ca2+li measured within the limits of our photometry based system. However, smal1 fluctuations were occasionally observed in the response of some individual cells (Fig. 5). Investigations by several groups suggest that intracellular stores of calcium are more important than extracellular calcium for the response to PGF,,. Pepperell et al. (1989) used rat luteal cells in suspension, and observed increased [Ca*+]i in response to PGF,,. This response to PGF,, was not eliminated by EGTA in the extracehular medium, but was decreased by dimethyl1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (CH 3-CH3-BAPTA) which chelates intracellular calcium. In other studies, an L calcium channel blocker, verapamil, had no effect on the inhibition of LH-stimulated CAMP formation by PGF,, (Lahav et al., 1983; Dorflinger et al., 1984). Radioactive calcium uptake studies show no effect of PGF,, on calcium uptake (Lahav et al., 1983). ~though Schwartz et al. (1989) have found T and L calcium channels on
142
chicken granulosa cells, a similar finding has not been reported in rat granulosa cells. In the present e~eriments, the involvement of extracellular and intracellular sources of calcium was investigated by administration of PGF,, during perifusion with low calcium EBSS. The magnitude of increase in [Ca*+], was diminished, and the response eventually eliminated over a period of approximately 10 min. Similarly, the [Ca*+]i response to GnRH was lost within 10 min during perifusion with low [Ca2+li media (Fig. 8). The magnitude of the response has been observed to gradually decrease over this time period as for PGF,, (unpublished obse~ations~. This supports the notion that the source of calcium involved in the increase in [Cazfli in response to PGF,, administration was intracellular, and that this intracellular pool was readily depleted. Furthermore, this study has demonstrated that simultaneous administration of GnRH and PGF,, caused an increase in [Ca2+li which was equal in magnitude to that produced by each agent alone. This suggests that the maximum amount of available calcium was released by either agent from the same source. The observation that shorter time intervals between PGF,, treatments decreased the magnitude of response suggests that the intracellular pool of releasable calcium can be depleted, and that these stores require a finite time for refilling. Alternatively, this observation may reflect a down-regulation of receptors, as postulated for a similar effect of GnRH in granulosa cells (Wang et al., 1989). The mechanism of receptor downregulation is suggested to involve internalization of membrane receptors into endocytic vesicles, and subsequent degradation of the GnRH-receptor complex (Hazum and Nimrod, 1982). GnRH and PGF,,, but not PGE,, stimulate PtdIns(4,5)P, breakdown in rat granulosa cells (Davis, 1984, 1986; Ma and Leung, 1985; Minegishi and Leung, 1985). The increase in [Ca2+li due to PGF,, and GnRH seen in these experiments is consistent with the release of calcium from intracellular stores by Ins(1,4,5)P,, which is a product of PtdIns(4,5)P, breakdown (Berridge et al., 1987). The lack of increase in [Ca2+li in response to PGE, is consistent with the absence of effect on PtdIns/PA labeling seen
even at high concentration of PGE, (Minegishi and Leung, 19851. In summa~, results of this study further support the view that the effects of PGF,, and of GnRH in rat granulosa cells are mediated by PtdIns(4,5)P, breakdown and a subsequent increase in [Ca2+li. The characteristics of this [Ca2+li response did not differ for the two agonists. Preliminary experiments indicate that the source of this [Ca2+], increase may be shared intracellular stores. References ~strong, D.T. (1981) J. Reprod. Fertii. 62, 283-291. Armstrong, D.T., Grinwich, D.L., Moon, Y.S. and Zamecnik, J. (1974) Life Sci. 14, 129-140. Bauminger, S. and Lindner, H.R. (1975) Prostaglandins 9, 737-751. Berridge, M.J. and Irvine, R.F. (1989) Nature 341, 197-205. Chang, J.P., Graeter, J. and Catt, K.J. (1986) Biochem. Biophys. Res. Commun. 134, 134-139. Clark, M.R. (1982) Endocrinology 110, 146-152. Corm, P.M., McArdle, CA., Andrew& W.V. and Huckle, W.R. (1987) Biol. Reprod. 36, 17-35. Davis, J.S., Farese, R.V. and Clark, M.R. (1983) Proc. Nat]. Acad. Sci. USA 80, 2049-2053. Davis, J.S., West, L.A. and Farese, R.V. (1984) Biochem. Biophys. Res. Commun. 122. 128991295. Davis, J.S., West, L.A. and Farese, R.V. (1986) Endocrinology 118, 2561-25’71. Dorflinger, L.J., Albert, P.J., Williams, A.T. and Behrman, H.R. (1984) Endocrinology 114, 1208-1215. Grynkiewicz, G., Poenie, M. and Tsien, Y. (1985) J. Biol. Chem. 260, 3440-3450. Hamberger, L., Janson, P.O. and Nilson, L. (1986) in Prostaglandins and their Inhibitors in Clinical Obstetrics and Gynecology (Bygdeman, M., Berger, G.S. and Keith, L.G., eds.), pp. 99-108, MTP Press, Boston, MA. Hazum, E. and Nimrod, A. (1982) Proc. Natl. Acad. Sci. USA 79(6), 1747- 1750. Hillensjo, T., LeMaire, W.J., Clark, M.R. and Ahren, L. (1982) Acta Endocrinol. 101, 603-610. Hsueh, A.J.W. and Erickson, G.F. (1979) Science 204, 854855. Hsueh, A.J.W. and Jones, P.C.W. (1981) Endocr. Rev. 2, 437-461. Hsueh, A.J.W., Adashi, E.Y., Jones, P.B.C. and Welsh, T.H. (1984) Endocr. Rev. 5, 76-127. Knecht, M., Ranta, T., Feng, P., Shinohara, 0. and Catt, K.J. (1985) J. Steroid Biochem. 23, 771-778. Labrie, F., Belanger, A., Seguin, C., Cusan, L., Pelletier, G., Lefebvre, F.A., Kelly, P.A., Ferland, L., Reeves, J.J., Lemay, A. and Raynaud, J.P. (1981) in Bioregulators of Reproduction (Jagiello, G. and Vogel, H.J., eds.), pp. 305-341, Academic Press, New York.
143 Lahav, M., Weiss, E., Rafaeloff, R. and Barzilai, D. (1983) J. Steroid Biochem. 19, 805-810. Lahav, M., West, L.A. and Davis, J.S. (1988) Endocrinology 123, 1044-1052. Lau, I.F., Saksena, SK. and Chang, M.C. (1974) J. Reprod. Fertil. 40, 467-469. LeMaire, W.J., Clark, M.R. and Marsh, J.M. (1979) in Human Ovulation (Hafez, E.S.E., ed.), pp. 159-175, Elsevier/ North-Holland, Amsterdam. Leung, P.C.K. (1985) Can. J. Physiol. Pharmacol. 63, 249-256. Leung, P.C.K., Raymond, V. and Labrie, F. (1983) Endocrinology 112, 1138-l 140. Ma, F. and Leung, P.C.K. (1985) Biochem. Biophys. Res. C’ommun. 130, 1201-1208. Minegishi, T. and Leung, P.C.K. (1985) Can. J. Physiol. Pharmacol. 63, 320-324. Naor, Z. and Yavin, E. (1982) Endocrinology 111, 1615-1619.
Nishizuka, Y. (1988) Nature 334, 661-665. Pepperell, J.R., Preston, S.L. and Behrman, H.R. (1989) Endocrinology 125, 144-151. Priddy, A.R. and Killick, S.R. (1988) Res. Reprod. 20, 3. Rodway, M.R., Baimbridge, K.G., Ho Yuen, B. and Leung, P.C.K. (1991) Endocrinology 129, 889-895 Schwartz, J.L., Asem, E.K., Mealing, G.A.R., Tsang, B.D., Rousseau, E.C., Whitfield, J.F. and Payet, M.D. (1989) Endocrinology 125, 1973-1982. Wang, J., Baimbridge, KG. and Leung, P.C.K. (1989) Endocrinology 124, 1912-1917. Watanabe, H., Tanaka, S., Akino, T. and Hasegawa-Sasaki, H. (1990) Biochem. Biophys. Res. Commun. 168, 328-334. Zor, U. and Lamprecht, S.A. (1977) in Biochemical Action of Hormones (Litwak, G., ed.), pp. 85-135, Academic Press, New York.
137
MOLCEL 02716
Prostaglandin F,, and gonadotropin-releasing hormone increase intracellular free calcium in rat granulosa cells Marie R. Rodway ‘, Gillian L. Steele a, Kenneth G. Bairnbridge b and Peter C.K. Leung ’ aDepartments of Obstetrics and Gynaecology and ’ Physiology, Uniuersity of British Columbia, Vancoucer, Canada (Received 12 September 1991; accepted 2 December 1991)
Key words: Prostaglandin F,,; Gonadotropin-releasing
hormone; Granulosa ceil; Calcium
Summary Changes in cytosolic free calcium concentration ([Ca2+]i) in response to prostaglandin F,, (PGF,,) and gonadotropin-releasing hormone (GnRH) were measured in single rat granulosa cells, using the calcium-sensitive fluorescent dye, fura-2AM. In 90 out of 135 granulosa cells (67%), there was a 3- to 4-fold increase in resting [Ca’+]i within 30 s of administration of PGF,, (10e6 M). The resting [Ca2+li returned to pre-stimulation levels in approximately 80 s. Granulosa cells were responsive to PGF,, at concentrations ranging from 10V7 M to lop4 M (n = 7). Within this range of concentrations, the magnitude of the calcium response did not differ. In another series of e~eriments, the majority (93%, n = 57) of the granuiosa cells which responded to PGF,, also responded to GnRH. Neither the magnitude of the [Ca2+li response nor the time to response differed between PGF,, and GnRH. Furthermore, simultaneous treatment of granulosa cells with both hormones did not generate a larger response than with either hormone alone. During perifusion with low calcium media, the characteristic [Ca”]i response to PGF,, decreased, and was eliminated within 10 min (n = 91. Similar observations were made in response to GnRH under these conditions. These data confirm that PGF,, and GnRH stimulate a transient increase in [Ca2+li in rat granulosa cells, the source of which may be shared intracellular stores.
Prostaglandin F,, (PGF,,) affects granulosa cell function and is believed to be involved in the
Correspondence to: Dr. Peter C.K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, Grace Hospital, 4490 Oak Street, Vancouver, B.C. V6H 3V5, Canada. This work was supported by grants from the Medical Research Council of Canada to K.G.B. and P.C.K.L.
mechanisms regulating ovulation (A~strong, 1981; Hsueh et al., 1984). Both the preovulatory Iuteinizing hormone (LH) surge and exogenous CAMP stimulate ovarian PG synthesis (Bauminger and Lindner, 1975; LeMaire et al., 1979) in follicles destined to ovulate (Hamberger et al., 1986). Indomethacin blocks this increase in PG synthesis at doses which prevent ovulation (Zor et al., 1977). The effect of indomethacin on ovulation can be reversed by injection of PGs, confirming the importance of PG production at this time (Hamberger et al., 1986; Priddy and Killick, 1988).
138
Furthermore, antisera to PGE, or PGF,, block LH-induced ovulation when administered systemically in mice (Lau et al., 1974), or intrafollicularly in estrous rabbits (Armstrong et al., 1974). In the latter study, antiserum to PGFzII was more effective than that of PGE, (Armstrong et al., 1974). In the rat, GnRH also exerts direct effects on ovarian function (Hsueh and Erickson, 1979; Hsueh and Jones, 1981; Labrie et al., 1981; Clark, 1982; Hillensjo et al., 1982; Knecht et al., 1985; Leung, 1985). Signal transduction of the growth hormone-releasing hormone (GnRH) message in the ovary, as in the pituitary (Chang et al., 1986; Conn et al., 1987), is believed to be via breakdown of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P,) (Naor and Yavin, 1982; Davis et al., 1983; Leung et al., 1983; Ma and Leung, 1985; Minegishi and Leung, 1985). The products of PtdIns(4,5)P, breakdown, diacylglycerol (DAG) and inositol 1,4,5_trisphosphate (Ins(1,4,5)P,), are second messengers which activate protein kinase C (PKC) and increase cytosolic free calcium ion concentrations ([Ca’+&), respectively (Nishizuka, 1988; Berridge and Irvine, 1989). We have previously demonstrated this elevation of [Ca2+li in response to GnRH in rat granulosa cells (Wang et al., 1989). Like GnRH, PGF,, enhances the turnover of phosphatidylinositol in rat granulosa cells (Minegishi and Leung, 19851, suggesting a similar transduction mechanism involving the metabolism of inositol phospholipids and calcium signalling. The purpose of these experiments was to investigate and compare the effects of PGF,, and GnRH on [Ca2+li in individual rat granulosa cells.
Materials
and methods
Animals
Immature Sprague-Dawley female rats (23 days old) were purchased from Charles River Canada (Montreal, Canada) and were injected S.C.with 12 IU pregnant mare’s serum gonadotropin. After 48 h, the rats were killed by cervical dislocation and granulosa ceils harvested as previously described (Minegishi and Leung, 1985).
Cell preparation
For intracellular calcium measurements, granulosa cells (0.1 X 105/ml) were plated in a monolayer onto 18 mm diameter uncoated glass coverslips and incubated in minimum essential medium (MEM; Gibco, Grand Island, NY, USA) containing 5% fetal bovine serum. After 48 h incubation at 37°C in an atmosphere of 5% CO, in air, cells were loaded with 5 PM fura-2-acetoxy-methyl ester (fura-2AM) (Molecular Probes, Eugene, OR, USA), in 1% dimethyl sulfoxide as previously described (Grynkiewicz et al., 1985; Wang et al., 1989). The coverslips were rinsed after 1 h incubation to wash out excess fura-2AM. Fluorescence measurement
Individual coverslips were mounted in a laminar flow-through chamber (volume approx. 350 ~1). Silicone rubber was used to complete a water-tight seal and the chamber inserted into a stainless steel holder. The assembly was then mounted onto the stage of a Zeiss Jenalumar microscope equipped for epifluorescence. The light source was a 200 Watt mercury arc lamp powered by a DC power supply. The light was first passed through one of three differential interference filters (350, 365, or 380 nm, bandwidths of 10 nm) mounted in a turret which could be rotated by a computer-controlled stepping motor. The light was then passed through a 410 nm dichroic mirror and a 100 x apochromat oil immersion lens with a numerical aperture of 1.4 and an adjustable diaphragm to reduce the light intensity. A field diaphragm in the light path prior to the dichroic mirror was used to reduce the area of illumination to the size of a single granulosa cell. All fluorescent light passed back through the dichroic mirror and a 450 nm band pass filter to reduce background fluorescence. The emitted fluorescence was deflected either to the eyepiece or to a camera port in which was mounted a photomultiplier tube, used to convert the fluorescence into a DC voltage. The voltage was converted to digital form and each ratio measurement was taken with an interval of 0.35 s and repeated either at 1.8 or 5 s intervals during hormone treatment. Between hormone treatments, resting [Ca2+li was determined approximately every 1 min to minimize exposure of the
cell to ultraviolet light. The method of measurement with our photomet~ based system would have precluded any observation of high frequency [Ca’+], oscihations, but slower oscillations (> 2 s) would have been observed. The responsiveness of the fura-2AM to changes in [Ca2+Ii was confirmed by direct injections into the laminar flow chamber of 500 PM Br-A23187, a non-fluorescent calcium ionophore (HSC Research Development Corp., Toronto, Canada). The cells could be used for up to 4-5 h after loading with only minimal signs of leakage of fura-2AM. Perifusion with Earle’s balanced salt solution (EBSS), or EBSS with no added calcium (designated low calcium EBSS) was at a rate of 4 mf/min. PGF,, and GnRH were administered directly into the chamber in 500 ~1 aliquots at a concentration of lop6 M. Hormones solubilized in EBSS were washed through with the perifusion medium except where otherwise indicated. Both PGF,, and GnRH were purchased from Sigma (St. Louis, MO, USA). Numbers are reported as mean rtr standard error of the mean. Student’s t-test was used to determine the significance of the difference between resting [Ca2+li in experiments where cells were perifused with low calcium EBSS or EBSS containing PGF,,. Results Changes in [Ca2+ji
4oo r-
-----
t
o
t
‘L__..L_--.._
1
-
.__!__J
0
20 l?ne
30
(min)
Fig. 1. The effect of PGF,, on [Ca2+li in a single rat granulosa ceil. Cells were prepared as described in Materials and methods. PGF,, was administered in a concentration of lo-’ M in 500 &I EBSS at times indicated by arrows in the figure. Similar results were seen in 90 individual cells in 25 experiments (n = 135).
Perifusion of granulosa cells with PGF,, (lo-’ MI in four separate experiments resulted in transient increases in [Ca*+l, similar to those seen with PGF,, administered in 500 ~1 aliquots. As shown in Fig. 3, the time from the start of perifusion until the cells responded was 1.5-2.3 min. The longer delay in response as compared to administration of 500 ~1 aliquots was likely due to the time required to fill the tubing and the chamber with PGF,,. The magnitude of the response was 4.6- to 7-fold, which was similar to the fold response to 500 ~1 aliquots of PGF,, (low6 M) administered in these cells. After 0.5-2.3 min, the [Ca’+]i fell to levels much lower than the
in response to PGF,,
A total of 135 granulosa cells were studied in 25 experiments. 67% of the cells responded to PGF;, (10e6 M) with an increase in [Ca*+J,. The average resting calcium level was 110 + 3.2 nM, the average response was 4.0 + 0.3-fold, the average time to response was 29 + 1.3 s, and to recovery was 82 + 7 s. A representative experiment is shown in Fig. 1. This response was not observed in cells treated with PGE, at low5 M (n = 16) or 1O-4 M (n = 18). The time between administrations of PGF?, was reduced in order to determine the minimum amount of time required to produce responses of equal magnitude. In four trials, the magnitude of the response began to diminish between 3 and 5 min (Fig. 2).
I
600
-10
0
10
20
--I 30
40
Time (mid
Fig. 2. The effect of a decrease in the time interval between administrations of PGF,, on alterations in ]Ca2+li in a single rat granuiosa cell. PGF,, (10m6 M in 500 ~1) was administered at times indicated by arrows in the figure. Similar results were seen in four cells.
140
PCS&
500
perifusion
’
800 GnRH
400 i
c ;;= N
600
_
400
_
h
200
-
2
2
;;+ 1
300
! -
*
200 I
0 100
.. 0,
Time (mid
,
X”
/ 0
Fig. 3. The effect of PGF,,
perifusion on [Ca2’li in a single rat granulosa cell. Cells were perifused with lo-’ M PGF,, (in EBSS) during the time period indicated by the black bar. The time delay to response of the cell was likely due to the time required to fill the tubing and chamber with media. Similar results were seen in four cells.
300
1
~------
t 0
to._
t
--_‘_tL$
t -
t
~-.-
20
-.
30
Fig. 4. An investigation of dose-response relationship of PGF,, in a single rat granulosa cell. PGF,, was administered in various concentrations at times indicated by the arrows in the figure. Seven individual cells produced similar results.
~
-_ ._-.-
Time (mid
Fig. 6. The effect of concurrent administration of GnRH and PGF,, on [Ca”’ Ii in a single rat granulosa cell. PGF,, and GnRH (IO-’ M in 500 ~1) were administered together, then separateiy to the same cell. Similar results were seen in five individual granulosa cells.
peak response, but significantly higher (p < 0.01) than the pre-perifusion resting [Ca2+],.
Time (mid
400
PGs*
i :
~_ll___--------
Investigation of concentration-response relationship and minimum effective concentration Granulosa cells responded to PGF,, with the characteristic increase in [Ca2+ji at concentrations ranging from lo-’ M to lop4 M. The magnitude of response in seven granulosa cells studied did not change when the concentration of PGF,, was increased (Fig. 4). Response to GnRH in cells responsive to PGF,, In another study of 84 granulosa cells, 53 cells (63%) responded to both GnRH and PGF?,; 22
-----
GnRH PGFu
300
no added
Ca2’
f 5 7 200 (u 3
100
0
.A”__L._I__
t 0
t, I
10
--
Time (mid
Fig. 5. The response of a single rat granulosa cell to GnRH and PGF,,. GnRH and PGF,, were administered at a concentration of lo-’ M in 500 JLI EBSS at times indicated by arrows in the figure. The number of cells responding to GnRH previously shown to be responsive to PGF,, was 53 out of 57 (93%). Note that the ICazcli response to PGF,, in this ceil shows small fluctuations.
0
10
Time (mid
Fig. 7. The effect of low calcium perifusion on the response of a single granulosa ceil to PGF,,. PGF,, was administered in a concentration of IO-’ M in 500 1.11EBSS at times indicated by arrows in the figure. Low calcium EBSS was perifused during the time indicated by the black bar. Nine cells responded in a similar fashion.
141
‘**~-1 5 S
h
i
no added Ca*’
t
t
0 0
I i0
6
Time (mini Fig. 8. The effect of low calcium perifusion on the response of a single granulosa cell to GnRH (n = 5). Cells were treated with the agonist (10e6 M in 500 ~1) at times indicated by the arrows. L,ow calcium medium was perifused during the time indicated by the black bar.
cells (26%) responded to GnRH only and four cells (5%) responded to PGF,, only; five (6%) did not respond to either agent. The average fold increase in [Ca2+li in response to GnRH was 3.0 I: 0.4, and the average time to response was 32.8 of:3.1 s (Fig. 5). ~~rn~i~e~ administration of PGF,,
and GnRH
In four experiments, granulosa celIs that responded to both PGF,, and GnRH (lo-” Ml showed no additive increase in [Ca*+], when both agents were administered in the same 500 pl aliquot of EBSS (Fig. 6, n = 9). Lonl calcium perifusion experiments Perifusion of granulosa cells with low calcium EBSS (n = 9) eliminated the [Ca2”li response to PGFZa in appro~mately 10 min (Fig. ‘7,p < 0.01). There was a gradual decrease in this [Ca2+li response over the perifusion period ~unpub~ished observations). The responsiveness of granulosa cells to GnRH was lost in a similar fashion over the same approximate time period following perifusion with low calcium media (Fig. 8, y1= 5). Discussion Using furamicrospectrofluorimetry, this study demonstrates an elevation of [Ca2+], in response to PGF,, in single rat granulosa cells. The response is similar to that reported for GnRH
in the same cell type; a rapid and transient one (Wang et al., 1989). The magnitude of the [Ca2’]i increase, latency and duration of response, as well as the nature of response were indisti~guishable between hormone treatments. There was no effect of increased PGF,, concentration on the magnitude of the response, as observed with GnRH. Furthermore, constant perifusion of the cells with either hormone resulted in a similar single and transient response (Wang et al., 1989). Interestingly, the majority (93%) of rat granulosa cells that responded to PGF,, with an increase in [Ca2+ji also responded to GnRH. Similar to the effects of GnRH on granulosa cells (Wang et al., 1989) and of PGF,, on luteal cells (Rodway et al., 1991) an interesting feature of the effect of PGF,, was that individual granulosa cells failed to demonstrate a dose response. Rather, these cells exhibited an all or none response to treatment with the prostaglandin. This would suggest that a dose-response effect seen when measurements are taken from a large population of cells reflects the recruitment of individual cells to the responding population, rather than each cell contributing more and more to the average [Ca”‘& signal. Further, we observed very littIe evidence that PGF,, consistently induced oscihations in [Ca2+li measured within the limits of our photometry based system. However, smal1 fluctuations were occasionally observed in the response of some individual cells (Fig. 5). Investigations by several groups suggest that intracellular stores of calcium are more important than extracellular calcium for the response to PGF,,. Pepperell et al. (1989) used rat luteal cells in suspension, and observed increased [Ca*+]i in response to PGF,,. This response to PGF,, was not eliminated by EGTA in the extracehular medium, but was decreased by dimethyl1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (CH 3-CH3-BAPTA) which chelates intracellular calcium. In other studies, an L calcium channel blocker, verapamil, had no effect on the inhibition of LH-stimulated CAMP formation by PGF,, (Lahav et al., 1983; Dorflinger et al., 1984). Radioactive calcium uptake studies show no effect of PGF,, on calcium uptake (Lahav et al., 1983). ~though Schwartz et al. (1989) have found T and L calcium channels on
142
chicken granulosa cells, a similar finding has not been reported in rat granulosa cells. In the present e~eriments, the involvement of extracellular and intracellular sources of calcium was investigated by administration of PGF,, during perifusion with low calcium EBSS. The magnitude of increase in [Ca*+], was diminished, and the response eventually eliminated over a period of approximately 10 min. Similarly, the [Ca*+]i response to GnRH was lost within 10 min during perifusion with low [Ca2+li media (Fig. 8). The magnitude of the response has been observed to gradually decrease over this time period as for PGF,, (unpublished obse~ations~. This supports the notion that the source of calcium involved in the increase in [Cazfli in response to PGF,, administration was intracellular, and that this intracellular pool was readily depleted. Furthermore, this study has demonstrated that simultaneous administration of GnRH and PGF,, caused an increase in [Ca2+li which was equal in magnitude to that produced by each agent alone. This suggests that the maximum amount of available calcium was released by either agent from the same source. The observation that shorter time intervals between PGF,, treatments decreased the magnitude of response suggests that the intracellular pool of releasable calcium can be depleted, and that these stores require a finite time for refilling. Alternatively, this observation may reflect a down-regulation of receptors, as postulated for a similar effect of GnRH in granulosa cells (Wang et al., 1989). The mechanism of receptor downregulation is suggested to involve internalization of membrane receptors into endocytic vesicles, and subsequent degradation of the GnRH-receptor complex (Hazum and Nimrod, 1982). GnRH and PGF,,, but not PGE,, stimulate PtdIns(4,5)P, breakdown in rat granulosa cells (Davis, 1984, 1986; Ma and Leung, 1985; Minegishi and Leung, 1985). The increase in [Ca2+li due to PGF,, and GnRH seen in these experiments is consistent with the release of calcium from intracellular stores by Ins(1,4,5)P,, which is a product of PtdIns(4,5)P, breakdown (Berridge et al., 1987). The lack of increase in [Ca2+li in response to PGE, is consistent with the absence of effect on PtdIns/PA labeling seen
even at high concentration of PGE, (Minegishi and Leung, 19851. In summa~, results of this study further support the view that the effects of PGF,, and of GnRH in rat granulosa cells are mediated by PtdIns(4,5)P, breakdown and a subsequent increase in [Ca2+li. The characteristics of this [Ca2+li response did not differ for the two agonists. Preliminary experiments indicate that the source of this [Ca2+], increase may be shared intracellular stores. References ~strong, D.T. (1981) J. Reprod. Fertii. 62, 283-291. Armstrong, D.T., Grinwich, D.L., Moon, Y.S. and Zamecnik, J. (1974) Life Sci. 14, 129-140. Bauminger, S. and Lindner, H.R. (1975) Prostaglandins 9, 737-751. Berridge, M.J. and Irvine, R.F. (1989) Nature 341, 197-205. Chang, J.P., Graeter, J. and Catt, K.J. (1986) Biochem. Biophys. Res. Commun. 134, 134-139. Clark, M.R. (1982) Endocrinology 110, 146-152. Corm, P.M., McArdle, CA., Andrew& W.V. and Huckle, W.R. (1987) Biol. Reprod. 36, 17-35. Davis, J.S., Farese, R.V. and Clark, M.R. (1983) Proc. Nat]. Acad. Sci. USA 80, 2049-2053. Davis, J.S., West, L.A. and Farese, R.V. (1984) Biochem. Biophys. Res. Commun. 122. 128991295. Davis, J.S., West, L.A. and Farese, R.V. (1986) Endocrinology 118, 2561-25’71. Dorflinger, L.J., Albert, P.J., Williams, A.T. and Behrman, H.R. (1984) Endocrinology 114, 1208-1215. Grynkiewicz, G., Poenie, M. and Tsien, Y. (1985) J. Biol. Chem. 260, 3440-3450. Hamberger, L., Janson, P.O. and Nilson, L. (1986) in Prostaglandins and their Inhibitors in Clinical Obstetrics and Gynecology (Bygdeman, M., Berger, G.S. and Keith, L.G., eds.), pp. 99-108, MTP Press, Boston, MA. Hazum, E. and Nimrod, A. (1982) Proc. Natl. Acad. Sci. USA 79(6), 1747- 1750. Hillensjo, T., LeMaire, W.J., Clark, M.R. and Ahren, L. (1982) Acta Endocrinol. 101, 603-610. Hsueh, A.J.W. and Erickson, G.F. (1979) Science 204, 854855. Hsueh, A.J.W. and Jones, P.C.W. (1981) Endocr. Rev. 2, 437-461. Hsueh, A.J.W., Adashi, E.Y., Jones, P.B.C. and Welsh, T.H. (1984) Endocr. Rev. 5, 76-127. Knecht, M., Ranta, T., Feng, P., Shinohara, 0. and Catt, K.J. (1985) J. Steroid Biochem. 23, 771-778. Labrie, F., Belanger, A., Seguin, C., Cusan, L., Pelletier, G., Lefebvre, F.A., Kelly, P.A., Ferland, L., Reeves, J.J., Lemay, A. and Raynaud, J.P. (1981) in Bioregulators of Reproduction (Jagiello, G. and Vogel, H.J., eds.), pp. 305-341, Academic Press, New York.
143 Lahav, M., Weiss, E., Rafaeloff, R. and Barzilai, D. (1983) J. Steroid Biochem. 19, 805-810. Lahav, M., West, L.A. and Davis, J.S. (1988) Endocrinology 123, 1044-1052. Lau, I.F., Saksena, SK. and Chang, M.C. (1974) J. Reprod. Fertil. 40, 467-469. LeMaire, W.J., Clark, M.R. and Marsh, J.M. (1979) in Human Ovulation (Hafez, E.S.E., ed.), pp. 159-175, Elsevier/ North-Holland, Amsterdam. Leung, P.C.K. (1985) Can. J. Physiol. Pharmacol. 63, 249-256. Leung, P.C.K., Raymond, V. and Labrie, F. (1983) Endocrinology 112, 1138-l 140. Ma, F. and Leung, P.C.K. (1985) Biochem. Biophys. Res. C’ommun. 130, 1201-1208. Minegishi, T. and Leung, P.C.K. (1985) Can. J. Physiol. Pharmacol. 63, 320-324. Naor, Z. and Yavin, E. (1982) Endocrinology 111, 1615-1619.
Nishizuka, Y. (1988) Nature 334, 661-665. Pepperell, J.R., Preston, S.L. and Behrman, H.R. (1989) Endocrinology 125, 144-151. Priddy, A.R. and Killick, S.R. (1988) Res. Reprod. 20, 3. Rodway, M.R., Baimbridge, K.G., Ho Yuen, B. and Leung, P.C.K. (1991) Endocrinology 129, 889-895 Schwartz, J.L., Asem, E.K., Mealing, G.A.R., Tsang, B.D., Rousseau, E.C., Whitfield, J.F. and Payet, M.D. (1989) Endocrinology 125, 1973-1982. Wang, J., Baimbridge, KG. and Leung, P.C.K. (1989) Endocrinology 124, 1912-1917. Watanabe, H., Tanaka, S., Akino, T. and Hasegawa-Sasaki, H. (1990) Biochem. Biophys. Res. Commun. 168, 328-334. Zor, U. and Lamprecht, S.A. (1977) in Biochemical Action of Hormones (Litwak, G., ed.), pp. 85-135, Academic Press, New York.
