485 Horm. Metab. Res. 11 (1979)
485~488
Effects of Epidermal Growth Factor, Fibroblast Growth Factor and Bovine Serum Albumin on Ornithine, Decarboxylase Activity of Porcine Granulosa Cells J. Osterman* and J.M. Hammond
Summary Epidermal growth factor, a potent mitrogen for granulosa ceUs produced a three-fold stimulation of ornithine decarboxylase activity in porcine granulose cells in vitro. Fibroblast growth factor, another compound with mitogenic activity for granulose cells, did not stimulate ornithine decarboxylase. Maximally effective concentrations of a commercial preparation of bovine serum albumin equalled the maximal effect of epidermal growth factor on this enzyme activity. The dominant stimulator(s) in the albumin preparation eluted after bovine serum albumin in gel filtration. At maximally effective concentrations, luteinizing hormone produced substantially greater stimulation than either epidermal growth factor or the bovine albumin preparation. Combinations of saturating doses of any two of these stimulators produced additive effects on enzyme activity. Key-Words: Omithine Decarboxylase Activity ~ Epidermal Gro.wth Factor ~ Fibroblast Growth Factor ~ Bovine Setum Albumin ~ Ovary
Sachs 1976). Consequently, the effect of these substanees on ovarian ODC aetivity was of interest. We have reeently shown a marked stimulation of this enzyme aetivity in poreine granulosa eells In vitro by luteinizing hormone (LH), follicle stimulating hormone (FSH) (Osterman and Hammond 1977) and prostaglandins of the E series (Osterman and Hammond 1978). Sinee this short-term culture system does not require serum, it was ideal for investigating the effeets of the polypeptide growth faetors on ODe. The eurrent study defines the effeets of EGF, FGF, and BSA on the ODC aetivity of poreine granulosa cells. The effeet of these agents has also been eompared to that of LH, a well-defined stimulator of ODe aetivity.
Materials and Methods Introduction Growth responses of various mammalian tissues are associated with marked stimulation of omithine deearboxylase (ODe), the rate-limiting enzyme of polyamine synthesis (RusseI 1973). Epidermal growth faetor (EGF) and fibroblast growth faetor (FGF) have been shown to dramatieally stimulate proliferation of ovarian eells in eulture and to reduee the eoneentration of serum neeessary for growth (Gospodarowicz, 111 and Birdwell 19773"; Gospodarowicz, 111 and Birdwell 1977b). Bovine serum albumin (BSA) has also been used as a serum substitute in maintaining granulose cells in eulture (Channing, Tsai and
·Current address: Department of Medicine, University of South Carolina School of Medicine, V.A. Hospital, Columbia, SC 29201, U.S.A.
Received: 29 Sept. 1978 0018-5043/79
0832-0485
Materillis. Ovine LH (NIH-LH-19) was obtained from the Hormone Distribution Office, National Institute of Arthritis, Metabolism and Digestive Diseases. EGF and FGF were purchased from Collaborative Research, Inc., Waltham, Mass. FGF was isolated from bovine pituitary gIands. Bovine serum albumin, Fraction V, was from Miles Laboratories, Inc. DL-( 1_14C) ornithine monohydrochloride (specific activity: 45 mCi/mmole) was purchased from New EngIand Nuclear. TisllUe preparation. Porcine ovaries were collected at a local slaughter house and the granulosa cells isolated from small (I ~2 mm) follicles. Approximately 10 8 cells/flask were incubated for 4 h in 10 ml serum-free Medium 199 as previously described (Osterman and Hammond 1977). LH (200 ng/mD and various concentrations of BSA, EGF, FGF were added at the beginning of incubation in respective experiments. This concentration of LH i~ supramaximal for stimulation of ODC (Osterman and Hammond 1977). Omithine Decarboxylase Assay. Following incubation granulosa cells were separated from the incubation medium, washed, homogenized and cytosol was prepared for the assay of ODC as previously described (Osterman and Hammond 1977). Each sampie was assayed in duplicate. The results are expressed as pmol/mg protein per 30 min. Total protein
Accepted: 28 Nov. 1978 S 03.00
© 1979 Georg Thieme Publishers
Downloaded by: National University of Singapore. Copyrighted material.
Department of Medicine, Division of Endocrinology, The Milton S. Hershey Medical Center, The Pennsylvania State University Hershey, Pennsylvania, U.S.A.
486
J. Ostennann, J.M. Hammond in the cytosol was detennined by the method of Lowry et a1. (1951). The precision of the culture technique and ODC assay has been characterized previously (Ostennan and Harn· mond 1977). In each experiment shown in the Results Se~ tion, granulosa cells were from a single pool of cells isolated on a particular day from 100-200 ovaries. Each experiment was repeated at least once with a different batch of ovaries and a similar pattern of response was obtained. Column Chromatography. BSA was fractionated on a I cm
0.5 2.5 10.020.0
0.5 20
+
+
+
Fig. I Effect of various concentrations of BSA, LH (200 ng/ml) and LH + BSA on ornithine decarboxylase activity of porcine granulosa cells. Results are means and range of detenninations on 2 replicate cultures
Preliminary studies showed the maximal effect of BSA on ODC activity to be between the 4th and 6th hour of incubation. In all experiments to be reported, granulosa cells were incubated for 4 h. The minimal concentration of BSA employed (0.5 mg/mi) caused more than a doubling of ODC activity and the plateau of activity was reached at 2.5 mg/ml (Fig. 1). BSA was a less effective stimulator of the enzyme activity than the saturating dose of LH. Saturating doses of BSA and LH in combination caused an effect which was more than additive.
2800 ~
~ _ 2400
~n
~ ~ 2000
~ " ~ ~ o .. 1600 :~
'"~ -5'" 1200 o E ~
0.
z~
800
Z
1!;
400
EGF (no /ml) LH (200 no/mi)
1.0 10.0100.0
1.0 10.0100.0
+
+ + +
Fig. 2 Effect of various concentrations of EGF, LH (200 ng/ml) and LH + EGF on ornitlline decarboxylase activity of porcine granulosa cells. Results are me ans and range of detenninations on 2 replicate cultures
...>->
0:-
)(
0
"...
800
.. '"
'" E a: ...... u ..
~~
...z -"
400
r
~
z
a: 0
FGF (no/mi) LH ( 200 119/ mI)
To determine if the ODC-stimulating activity of the BSA preparation employed resided in bovine serum albumin per se, we fractionated this preparation on a calibrated Sephadex G-200 column. More than 80% of the activity in this preparation eluted after serum albumin. The activity of the low molecular weight stimulator(s) per mg protein was more than 1000-fold greater than that of the parent BSA preparation. Figure 2 shows that the minimally effective dose of EGF was 1.0 ng/ml; the dose of 10 ng/ml caused approximately a three-fold stimulation of the enzyme activity whereas the 100 ng/ml dose was slightly less effective. LH was again a much more effective stimulator of the enzyme activity than EGF. The combination of EGF and LH again produced an effect which was more than additive. In contrast to EGF, FGF in the dose range (0.1-10 ng/ml) caused no stimulation of ODC activity and was possibly slightly inhibitory (Fig. 3).
1200
~ .~ ~~ ~ .. " >-
Results
0.1
1.0 10.0
0.1
+
+
1.0 10.0
+
+
Fig. 3 Effect of various concentrations of FGF, LH (200 ngiml) and LH + EGF on ornithine decarboxylase activity of porcine granulosa cells. Results are means and range of detenninations on 2 replicate cultures
Figure 4 shows that the combination of maxima\1y effective concentrations of EGF and bovine serum albumin had an additive effect in stimulation of ODe activity.
Downloaded by: National University of Singapore. Copyrighted material.
8SA (mo/mi) LH (200 no/mi)
x 100 cm column of Sephadex G-200 (Pharmacia Fine Chemicals), equilibrated and eluted in .25 M KCI, 20 mM Tris buffer, pH 7.4 The BSA peak was quantitated by protein determinations (Lowry et aL 1951), pooled, and concentrated in a filtration cell using a membrane with a 1500 dalton exclusion limit (Amicon, Inc.). The retentate was anaIyzed for pro tein content (Lowry et a1. 195 I), and added to granulosa cells to test its stimulation of ODC activity. The material eluting between the tall of the BSA peak and the salt volume was handled similarly.
Effect of Epidennal and Fibroblast Growth Factors on Ornithine Decarboxylase Activity of Porcine Granulose Cells 487
1600 ~ :;
;:: ~ E1200
.... E
Sg >~
x .. oe. CI> .., ~ ~ 800
....
(.)
..'"
°0
.... E
z e. x-
~ CI: o
400
CONTROL BSA
EGF
BSA+EGF
Fig. 4 Effect of saturating concentrations of BSA (2.5 mgl ml), EGF (10 ng/m!) and thcir combination on ornithine decarboxylase activity of porcine granulosa cells. Results are mean ± SEM of detenninations on 3 replicate cultures
Discussion This study indieates definite stimulation of the OOC activity of porcine granulosa cells from small follides by EGF but not FGF in the dose range ernployed. Although both FGF and EGF were mitogenie for bovine granulosa eells (Gospodarowicz, 111 and Birdwell 1977a), their relative aetivity apparentIy shifted with luteinization: EGF was a more patent mitogen for granulosa cells from medium-sized bovine follides (Gospodarowicz, III and Birdwell 1977a), whereas with luteinized cells, FGF was a potent mitogen and EGF was essentially inactive (Gospodarowicz, 111 and Birdwell 1977b). The response of the immature granulosa cells employed for the current studies may represent a still earlier stage in differentiation before responsiveness to FGF is attained. In these studies, saturating concentrations of a BSA preparation were as effective as EGF in stimulating ODe activity. Our preliminary fractionation studies indicate that most of this activity can be accounted for by contaminating substances of smaller molecular size. These results are thus reminiseent of the findings of Morley with fractionated serum and hepatic ODe (Morley 1972; Morley 1974). It is dear that commercial BSA preparations cannot be used as serum substitutes if medium is to remain truly "defined". Quantities of the stimulating material obtained to date have not permitted determination of moleeular weight, chemieal nature, or precise biological potency. The active substance(s) does not seem to be EGF per se, since the effects of EGE were additive to those of BSA at saturating concentrations of each. Similarly additive effects were seen when
The current data are consistent with the involvement of OOC and polyamines in EGF·induced granulosa cell replication. It is not dear, however, that ODC stimulation is related to granulosa cell proliferation in an exdusive or ob liga tory fashion. Thus, the concentrations of EGF required for ODe stimulation in our studies appear substantially higher than those required for enhancement of DNA synthesis by bovine granulosa cells (Gospodarowicz, III and Birdwell 1977a). In addition, the magnitude of the maximal OOC response to EGF or BSA reported here is small when compared to the stimulation caused by EGF in chick embryo cells (Stastny and Cohen 1970), or by FSH, LH (Ostennan and Hammond 1977), prostaglandins (Ostennan and Hammon.d 1978), or cAMP analogues (Ostennan, Demers and Hammond 1978) in our previous studies with cuItured granulosa cells. With the exception of FSH (Thanki and Channing 1976; McNatty and Sawyers 1975), these potent stimulators of ODe activity in granulosa cells have not been shown to be mitogenic for these cells in vitro. LH has an anti-mitotic effect on granulosa cells Thanki and Clumning 1976; McNatty and Sawyers 1975); with luteal cells, LH, dibutyryl cAMP, and prostaglandins have all been found to suppress mitosis (Gospodarowicz and Gospodarowicz 1975). However, all of these agents eause impressive increases in steroidogenesis (Channing and Tsa!riri 1977; Thanki and Channing 1978). Thus, the available evidence appears to link ODC more strongly with differentiated function in the granulosa cell than with the proliferative response of these eells. Acknow1edgements The authors wish to acknowledge the help of Dr. Gien Gunsalus who helped with the chromatography, the Hormone Distribution Program, NIAMDD, for ovine LH, Ms. Elizabeth Krall for technical assistance, and Mrs. Marlene Thompson and Mrs. Ann Martin for secretarial services. Supported by NIH Grant No. HD) 0) 22
Re!erences Channing, c.P., A. Tsafriri: Mechanism of action of lutein-
izing honnone and follicle-stimulating hormone on the ovary in vitro. Metabolism 26: 413-468(1977) Channing, c.P., V. Tsai, D. Sachs: Role of insulin, thyroxin and cortisol in lu teinization of porcine granulosa cells grown in chemically defined media. Biol. Reprod. 15: 233-247 (1976) Gospodarowicz, D., F. Gospodarowicz: The morphological transfonnation and inhibition of growth of bovine luteal ceIJs in tissue culture induced by luteinizing hormone and dibutyryl cyclic AMP. Endocrinology 96: 438-467 (1975)
Downloaded by: National University of Singapore. Copyrighted material.
LH was combined with either EGF or BSA. These results suggest a discrete action for each of the stimulators evaluated.
M. Hamaji, K. Nakao, K. Kiso
Gospodarowicz, D., c'R. lll, c'R. Birdwell: Effects of fibr()o blast and epidermal growth factors on ovarian cell pr()o liferation in vitro. I. Characterization of the response of grnulosa cells to FGF and EGF. Endocrinology 100: 1108-1120 (I 977a) Gospodarowicz, D., c'R. fll, c'R. Birdwell: Effects of fibr()o blast and epidermal growth factors on ovarian cell pr()o liferation in vitro. 11. Proliferative response of luteal cells to FGF but not EGF. Endocrinology 100: 11211128 (I 977b) Lowry. O.H., N.J. Rosebrough. A.L. Farr. R.J. RandDll: Pro tein measurement with the folin phenol reagent J. BioL Chem. 193: 265-275 (1951) McNatty. K.P., R.S. Sawyers: Relationship between the endocrine environment within the Graafian follicle and the subsequent rate of progesterone secretion by human granulosa cells in vitro. J. Endocrinol. 66: 391-400 (1975) Morley, CD. G.: The stimulation of liver ornithine decar~ oxylase by a factor from fetal calf serum. Biochem. Bi()o phys. Res. Commun. 49: 1530-1535 (1972) Morley, c'D.G.: The regulation of cell growth. 11. Some characteristics of a fetal calf· serum (FF 2) stimulating ornithine decarboxylase in mouse liver. Biochim. Bi()o phys. Acta 362: 480-492 (1974)
OstermJUl. J., J.M. Hammond: FSH and LH stimulation of ornithine decarboxylase activity: Studies with porcine granulosa ceUs in vitro. Endocrinology 101: 1335-1338 (1977) Osterman, J., J.M. Hammond: Prostagiandin stimulation of ovarian ornithine decarboxylase in vitro. Biochem. Bi()o phys. Res. Commun. 83: 794-799 (1978) Osterman, J., L.M. Demers. J.M. Hammond: Gonadotropin stimulation of porcine ovarian ornithine decarboxylase in vitro: The role of cyclic AMP. Endocrinology 103: 1718-1724 (1978) Russel. D.H.: Polyamines in growth - normal and neopl
485~488
Effects of Epidermal Growth Factor, Fibroblast Growth Factor and Bovine Serum Albumin on Ornithine, Decarboxylase Activity of Porcine Granulosa Cells J. Osterman* and J.M. Hammond
Summary Epidermal growth factor, a potent mitrogen for granulosa ceUs produced a three-fold stimulation of ornithine decarboxylase activity in porcine granulose cells in vitro. Fibroblast growth factor, another compound with mitogenic activity for granulose cells, did not stimulate ornithine decarboxylase. Maximally effective concentrations of a commercial preparation of bovine serum albumin equalled the maximal effect of epidermal growth factor on this enzyme activity. The dominant stimulator(s) in the albumin preparation eluted after bovine serum albumin in gel filtration. At maximally effective concentrations, luteinizing hormone produced substantially greater stimulation than either epidermal growth factor or the bovine albumin preparation. Combinations of saturating doses of any two of these stimulators produced additive effects on enzyme activity. Key-Words: Omithine Decarboxylase Activity ~ Epidermal Gro.wth Factor ~ Fibroblast Growth Factor ~ Bovine Setum Albumin ~ Ovary
Sachs 1976). Consequently, the effect of these substanees on ovarian ODC aetivity was of interest. We have reeently shown a marked stimulation of this enzyme aetivity in poreine granulosa eells In vitro by luteinizing hormone (LH), follicle stimulating hormone (FSH) (Osterman and Hammond 1977) and prostaglandins of the E series (Osterman and Hammond 1978). Sinee this short-term culture system does not require serum, it was ideal for investigating the effeets of the polypeptide growth faetors on ODe. The eurrent study defines the effeets of EGF, FGF, and BSA on the ODC aetivity of poreine granulosa cells. The effeet of these agents has also been eompared to that of LH, a well-defined stimulator of ODe aetivity.
Materials and Methods Introduction Growth responses of various mammalian tissues are associated with marked stimulation of omithine deearboxylase (ODe), the rate-limiting enzyme of polyamine synthesis (RusseI 1973). Epidermal growth faetor (EGF) and fibroblast growth faetor (FGF) have been shown to dramatieally stimulate proliferation of ovarian eells in eulture and to reduee the eoneentration of serum neeessary for growth (Gospodarowicz, 111 and Birdwell 19773"; Gospodarowicz, 111 and Birdwell 1977b). Bovine serum albumin (BSA) has also been used as a serum substitute in maintaining granulose cells in eulture (Channing, Tsai and
·Current address: Department of Medicine, University of South Carolina School of Medicine, V.A. Hospital, Columbia, SC 29201, U.S.A.
Received: 29 Sept. 1978 0018-5043/79
0832-0485
Materillis. Ovine LH (NIH-LH-19) was obtained from the Hormone Distribution Office, National Institute of Arthritis, Metabolism and Digestive Diseases. EGF and FGF were purchased from Collaborative Research, Inc., Waltham, Mass. FGF was isolated from bovine pituitary gIands. Bovine serum albumin, Fraction V, was from Miles Laboratories, Inc. DL-( 1_14C) ornithine monohydrochloride (specific activity: 45 mCi/mmole) was purchased from New EngIand Nuclear. TisllUe preparation. Porcine ovaries were collected at a local slaughter house and the granulosa cells isolated from small (I ~2 mm) follicles. Approximately 10 8 cells/flask were incubated for 4 h in 10 ml serum-free Medium 199 as previously described (Osterman and Hammond 1977). LH (200 ng/mD and various concentrations of BSA, EGF, FGF were added at the beginning of incubation in respective experiments. This concentration of LH i~ supramaximal for stimulation of ODC (Osterman and Hammond 1977). Omithine Decarboxylase Assay. Following incubation granulosa cells were separated from the incubation medium, washed, homogenized and cytosol was prepared for the assay of ODC as previously described (Osterman and Hammond 1977). Each sampie was assayed in duplicate. The results are expressed as pmol/mg protein per 30 min. Total protein
Accepted: 28 Nov. 1978 S 03.00
© 1979 Georg Thieme Publishers
Downloaded by: National University of Singapore. Copyrighted material.
Department of Medicine, Division of Endocrinology, The Milton S. Hershey Medical Center, The Pennsylvania State University Hershey, Pennsylvania, U.S.A.
486
J. Ostennann, J.M. Hammond in the cytosol was detennined by the method of Lowry et a1. (1951). The precision of the culture technique and ODC assay has been characterized previously (Ostennan and Harn· mond 1977). In each experiment shown in the Results Se~ tion, granulosa cells were from a single pool of cells isolated on a particular day from 100-200 ovaries. Each experiment was repeated at least once with a different batch of ovaries and a similar pattern of response was obtained. Column Chromatography. BSA was fractionated on a I cm
0.5 2.5 10.020.0
0.5 20
+
+
+
Fig. I Effect of various concentrations of BSA, LH (200 ng/ml) and LH + BSA on ornithine decarboxylase activity of porcine granulosa cells. Results are means and range of detenninations on 2 replicate cultures
Preliminary studies showed the maximal effect of BSA on ODC activity to be between the 4th and 6th hour of incubation. In all experiments to be reported, granulosa cells were incubated for 4 h. The minimal concentration of BSA employed (0.5 mg/mi) caused more than a doubling of ODC activity and the plateau of activity was reached at 2.5 mg/ml (Fig. 1). BSA was a less effective stimulator of the enzyme activity than the saturating dose of LH. Saturating doses of BSA and LH in combination caused an effect which was more than additive.
2800 ~
~ _ 2400
~n
~ ~ 2000
~ " ~ ~ o .. 1600 :~
'"~ -5'" 1200 o E ~
0.
z~
800
Z
1!;
400
EGF (no /ml) LH (200 no/mi)
1.0 10.0100.0
1.0 10.0100.0
+
+ + +
Fig. 2 Effect of various concentrations of EGF, LH (200 ng/ml) and LH + EGF on ornitlline decarboxylase activity of porcine granulosa cells. Results are me ans and range of detenninations on 2 replicate cultures
...>->
0:-
)(
0
"...
800
.. '"
'" E a: ...... u ..
~~
...z -"
400
r
~
z
a: 0
FGF (no/mi) LH ( 200 119/ mI)
To determine if the ODC-stimulating activity of the BSA preparation employed resided in bovine serum albumin per se, we fractionated this preparation on a calibrated Sephadex G-200 column. More than 80% of the activity in this preparation eluted after serum albumin. The activity of the low molecular weight stimulator(s) per mg protein was more than 1000-fold greater than that of the parent BSA preparation. Figure 2 shows that the minimally effective dose of EGF was 1.0 ng/ml; the dose of 10 ng/ml caused approximately a three-fold stimulation of the enzyme activity whereas the 100 ng/ml dose was slightly less effective. LH was again a much more effective stimulator of the enzyme activity than EGF. The combination of EGF and LH again produced an effect which was more than additive. In contrast to EGF, FGF in the dose range (0.1-10 ng/ml) caused no stimulation of ODC activity and was possibly slightly inhibitory (Fig. 3).
1200
~ .~ ~~ ~ .. " >-
Results
0.1
1.0 10.0
0.1
+
+
1.0 10.0
+
+
Fig. 3 Effect of various concentrations of FGF, LH (200 ngiml) and LH + EGF on ornithine decarboxylase activity of porcine granulosa cells. Results are means and range of detenninations on 2 replicate cultures
Figure 4 shows that the combination of maxima\1y effective concentrations of EGF and bovine serum albumin had an additive effect in stimulation of ODe activity.
Downloaded by: National University of Singapore. Copyrighted material.
8SA (mo/mi) LH (200 no/mi)
x 100 cm column of Sephadex G-200 (Pharmacia Fine Chemicals), equilibrated and eluted in .25 M KCI, 20 mM Tris buffer, pH 7.4 The BSA peak was quantitated by protein determinations (Lowry et aL 1951), pooled, and concentrated in a filtration cell using a membrane with a 1500 dalton exclusion limit (Amicon, Inc.). The retentate was anaIyzed for pro tein content (Lowry et a1. 195 I), and added to granulosa cells to test its stimulation of ODC activity. The material eluting between the tall of the BSA peak and the salt volume was handled similarly.
Effect of Epidennal and Fibroblast Growth Factors on Ornithine Decarboxylase Activity of Porcine Granulose Cells 487
1600 ~ :;
;:: ~ E1200
.... E
Sg >~
x .. oe. CI> .., ~ ~ 800
....
(.)
..'"
°0
.... E
z e. x-
~ CI: o
400
CONTROL BSA
EGF
BSA+EGF
Fig. 4 Effect of saturating concentrations of BSA (2.5 mgl ml), EGF (10 ng/m!) and thcir combination on ornithine decarboxylase activity of porcine granulosa cells. Results are mean ± SEM of detenninations on 3 replicate cultures
Discussion This study indieates definite stimulation of the OOC activity of porcine granulosa cells from small follides by EGF but not FGF in the dose range ernployed. Although both FGF and EGF were mitogenie for bovine granulosa eells (Gospodarowicz, 111 and Birdwell 1977a), their relative aetivity apparentIy shifted with luteinization: EGF was a more patent mitogen for granulosa cells from medium-sized bovine follides (Gospodarowicz, III and Birdwell 1977a), whereas with luteinized cells, FGF was a potent mitogen and EGF was essentially inactive (Gospodarowicz, 111 and Birdwell 1977b). The response of the immature granulosa cells employed for the current studies may represent a still earlier stage in differentiation before responsiveness to FGF is attained. In these studies, saturating concentrations of a BSA preparation were as effective as EGF in stimulating ODe activity. Our preliminary fractionation studies indicate that most of this activity can be accounted for by contaminating substances of smaller molecular size. These results are thus reminiseent of the findings of Morley with fractionated serum and hepatic ODe (Morley 1972; Morley 1974). It is dear that commercial BSA preparations cannot be used as serum substitutes if medium is to remain truly "defined". Quantities of the stimulating material obtained to date have not permitted determination of moleeular weight, chemieal nature, or precise biological potency. The active substance(s) does not seem to be EGF per se, since the effects of EGE were additive to those of BSA at saturating concentrations of each. Similarly additive effects were seen when
The current data are consistent with the involvement of OOC and polyamines in EGF·induced granulosa cell replication. It is not dear, however, that ODC stimulation is related to granulosa cell proliferation in an exdusive or ob liga tory fashion. Thus, the concentrations of EGF required for ODe stimulation in our studies appear substantially higher than those required for enhancement of DNA synthesis by bovine granulosa cells (Gospodarowicz, III and Birdwell 1977a). In addition, the magnitude of the maximal OOC response to EGF or BSA reported here is small when compared to the stimulation caused by EGF in chick embryo cells (Stastny and Cohen 1970), or by FSH, LH (Ostennan and Hammond 1977), prostaglandins (Ostennan and Hammon.d 1978), or cAMP analogues (Ostennan, Demers and Hammond 1978) in our previous studies with cuItured granulosa cells. With the exception of FSH (Thanki and Channing 1976; McNatty and Sawyers 1975), these potent stimulators of ODe activity in granulosa cells have not been shown to be mitogenic for these cells in vitro. LH has an anti-mitotic effect on granulosa cells Thanki and Clumning 1976; McNatty and Sawyers 1975); with luteal cells, LH, dibutyryl cAMP, and prostaglandins have all been found to suppress mitosis (Gospodarowicz and Gospodarowicz 1975). However, all of these agents eause impressive increases in steroidogenesis (Channing and Tsa!riri 1977; Thanki and Channing 1978). Thus, the available evidence appears to link ODC more strongly with differentiated function in the granulosa cell than with the proliferative response of these eells. Acknow1edgements The authors wish to acknowledge the help of Dr. Gien Gunsalus who helped with the chromatography, the Hormone Distribution Program, NIAMDD, for ovine LH, Ms. Elizabeth Krall for technical assistance, and Mrs. Marlene Thompson and Mrs. Ann Martin for secretarial services. Supported by NIH Grant No. HD) 0) 22
Re!erences Channing, c.P., A. Tsafriri: Mechanism of action of lutein-
izing honnone and follicle-stimulating hormone on the ovary in vitro. Metabolism 26: 413-468(1977) Channing, c.P., V. Tsai, D. Sachs: Role of insulin, thyroxin and cortisol in lu teinization of porcine granulosa cells grown in chemically defined media. Biol. Reprod. 15: 233-247 (1976) Gospodarowicz, D., F. Gospodarowicz: The morphological transfonnation and inhibition of growth of bovine luteal ceIJs in tissue culture induced by luteinizing hormone and dibutyryl cyclic AMP. Endocrinology 96: 438-467 (1975)
Downloaded by: National University of Singapore. Copyrighted material.
LH was combined with either EGF or BSA. These results suggest a discrete action for each of the stimulators evaluated.
M. Hamaji, K. Nakao, K. Kiso
Gospodarowicz, D., c'R. lll, c'R. Birdwell: Effects of fibr()o blast and epidermal growth factors on ovarian cell pr()o liferation in vitro. I. Characterization of the response of grnulosa cells to FGF and EGF. Endocrinology 100: 1108-1120 (I 977a) Gospodarowicz, D., c'R. fll, c'R. Birdwell: Effects of fibr()o blast and epidermal growth factors on ovarian cell pr()o liferation in vitro. 11. Proliferative response of luteal cells to FGF but not EGF. Endocrinology 100: 11211128 (I 977b) Lowry. O.H., N.J. Rosebrough. A.L. Farr. R.J. RandDll: Pro tein measurement with the folin phenol reagent J. BioL Chem. 193: 265-275 (1951) McNatty. K.P., R.S. Sawyers: Relationship between the endocrine environment within the Graafian follicle and the subsequent rate of progesterone secretion by human granulosa cells in vitro. J. Endocrinol. 66: 391-400 (1975) Morley, CD. G.: The stimulation of liver ornithine decar~ oxylase by a factor from fetal calf serum. Biochem. Bi()o phys. Res. Commun. 49: 1530-1535 (1972) Morley, c'D.G.: The regulation of cell growth. 11. Some characteristics of a fetal calf· serum (FF 2) stimulating ornithine decarboxylase in mouse liver. Biochim. Bi()o phys. Acta 362: 480-492 (1974)
OstermJUl. J., J.M. Hammond: FSH and LH stimulation of ornithine decarboxylase activity: Studies with porcine granulosa ceUs in vitro. Endocrinology 101: 1335-1338 (1977) Osterman, J., J.M. Hammond: Prostagiandin stimulation of ovarian ornithine decarboxylase in vitro. Biochem. Bi()o phys. Res. Commun. 83: 794-799 (1978) Osterman, J., L.M. Demers. J.M. Hammond: Gonadotropin stimulation of porcine ovarian ornithine decarboxylase in vitro: The role of cyclic AMP. Endocrinology 103: 1718-1724 (1978) Russel. D.H.: Polyamines in growth - normal and neopl
