COMPARISON OF THE STIMULATORY EFFECTS OF OVINE, PORCINE AND HUMAN FOLLICLE-STIMULATING HORMONE
AND OF OVINE AND HUMAN LUTEINIZING HORMONE ON THE ACCUMULATION OF CYCLIC AMP BY PORCINE GRANULOSA CELLS CORNELIA P. CHANNING Department of Physiology, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, Maryland21201, U.S.A. ADA M. LINDSEY
AND
(Received 11 January 1978) SUMMARY
The effects of ovine, porcine and human FSH, and ovine and human LH on the accumulation of cyclic AMP by porcine granulosa cells obtained from follicles at various stages of maturation were investigated. During incubation periods of 15 min, 10 \g=m\g ovine FSH pretreated with antiserum to LH or 10 \g=m\ghuman FSH resulted in an 11- to 18-fold, five\x=req-\ to ninefold, and less than a twofold increase in intracellular accumulation of cyclic AMP by granulosa cells from small (1\p=n-\2 mm), medium (3\p=n-\5 mm) and large (6\p=n-\12 mm) follicles respectively. Similar patterns of response occurred with addition of porcine FSH. After incubation for 30 and 60 min with ovine, porcine or human FSH, significant accumulation of cyclic AMP in the incubation medium occurred with cells obtained from small and medium-sized follicles. After 60 min of incubation with FSH the accumulation of cyclic AMP in the incubation medium exceeded the intracellular cyclic AMP levels in granulosa cells from small and medium-sized follicles. During incubation periods of 15 min, 1\m=.\0\g=m\govine LH resulted in less than a twofold, a fourfold and greater than a tenfold increase in intracellular accumulation of cyclic AMP by granulosa cells from small, medium and large follicles respectively. Addition of human LH brought about a similar response. Incubation periods of 30 and 60 min with 1\m=.\0 \g=m\g ovine or human LH resulted in significant accumulation of cyclic AMP in the incubation medium by granulosa cells from large follicles; cyclic AMP content in the incubation medium was greater after 60 min compared with 30 min of incubation. It was concluded that ovine FSH pretreated with an antiserum to LH had similar effects on cyclic AMP levels as did purified human and porcine FSH, and that the stimulatory effects of the less pure ovine FSH were probably not due to an impurity in the FSH preparation. Porcine granulosa cells obtained from small follicles should be suitable as an in-vitro FSH bioassay while granulosa cells obtained from large follicles should be suitable as an in-vitro LH bioassay. INTRODUCTION
Gonadotrophin-mediated ovarian responses depend on not only the concentration of biologically active hormone but also the responsiveness of the target cell. It is generally believed that the first step in the mechanism of action of these glycoprotein hormones is their interaction with specific receptors in the target cells, which subsequently stimulates the formation of cyclic AMP from ATP (Robinson, Butcher & Sutherland, 1971). The cyclic AMP thus generated leads to biological effects on cellular metabolism and steroido¬ genesis and has been designated the second messenger of hormone action.
previously that ovine follicle-stimulating hormone (FSH) against luteinizing hormone (LH) has a greater stimulatory effect on the accumulation of cyclic AMP in cells obtained from small and medium follicles than in cells from large follicles (Lindsey & Channing, 1976). Since the ovine FSH pre¬ paration was not highly purified, the possibility existed that an impurity therein might be responsible for its action. To rule out this possibility, the effect of the less pure ovine FSH was compared with the effect of more highly purified FSH preparations from porcine and human sources. The effect of LH on granulosa cells in previous studies had been examined by measuring only the accumulation of cyclic AMP within the cells. In the present studies, measurement of the accumulation of cyclic AMP within the cells and in the incubation medium was used to examine the stimulatory effects of the gonadotrophins on porcine granulosa cells from small, medium and large follicles. The experiments were also designed to determine whether there were any differences in the response of porcine granulosa cells to gonadotrophins obtained from porcine, human and ovine sources. It has been demonstrated pretreated with an antiserum
MATERIALS AND METHODS
Granulosa cells Porcine ovaries were obtained within 15 min of death at a local meat-packing factory and were transported to the laboratory in ice-cold 0-9% NaCl solution containing 100 units penicillin and 100 µg streptomycin/ml. Granulosa cells were obtained from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles as described by Channing & Ledwitz-Rigby (1975). Granulosa cells were separated from follicular fluid by centrifugation at 600 g for 5 min and were washed with Eagle's medium three times. After resuspension in Eagle's medium (pH 7-4; Grand Island Biological Co., Grand Island, New York, U.S.A.) plus 1% bovine serum albumin (BSA) fraction V (Sigma Chemical Company), the granulosa cells were kept overnight at 4 °C until 1 h before use when they were allowed to equilibrate with room temperature. With dye exclusion as an indication of cell viability, the cells were counted in a haemocytometer in 0-06% trypan blue. Hormones and chemicals 1-10 i.u. NIH-oFSH-Sl/mg) and ovine LH
Ovine FSH (NIH-oFSH-SlO; (NIH-0LH-SI8; 1-03 i.u. NIH-oLH-Sl/mg) were used. The ovine FSH preparation contained less than 0-01 i.u. NIH-LH-Sl/mg and the ovine LH less than 0-05 i.u. NIH-FSH-Sl/mg. The potencies of the FSH and LH preparations were determined by the Steelman-Pohley bioassay (Steelman & Pohley, 1953) and the ovarian ascorbic acid depletion assay (Parlow, 1961) respectively. These hormones were supplied by the Hormone Distribution Officer of the National Institute of Arthritis and Metabolic and Digestive Diseases, NIH. Antiserum to bovine LH, generated in a mare (Snook, 1968), was donated by Dr William Hansel of Cornell University, and was used to neutralize the LH contaminant in the ovine FSH preparation by the following procedure. Various concentrations of ovine FSH and LH were pre-incubated with the antiserum to LH for 1 h at 37 °C before the granulosa cells were added. Antiserum dilutions 1 : 500 to 1 : 5000 were used depending on the concen¬ tration of LH to be neutralized. The dilutions of antiserum were determined experimentally; estimates of LH contaminating the ovine FSH preparation were calculated and ovine LH was pre-incubated with the antiserum to LH to demonstrate that the antiserum was effective at neutralizing the stimulatory effect of LH on granulosa cell accumulation of cyclic AMP. We have shown previously (Lindsey & Channing, 1976, 1978) that pre-incubation of ovine FSH with antiserum to LH can neutralize the ability of contaminating LH to stimulate the release of cyclic AMP in porcine granulosa cells. Therefore, in all the present experiments, ovine FSH was pretreated with antiserum to LH.
Highly purified human LH (LER-1705, potency 3800 i.u./mg and FSH activity 3 i.u./mg) and human FSH (LER-1577c, potency 880 i.u./mg) were provided by Dr L. E. Reichert, Jr. The potencies of human FSH and LH are expressed in terms of the Second International Reference Preparation of Human Menopausal Gonadotrophin Standard (2nd IRP-hMG standard). The FSH preparation had been pretreated with chymotrypsin to inactivate selectively the contaminating LH (Reichert, 1967); the residual LH activity in the FSH preparation had a potency of 5-7 i.u./mg. The resistance of FSH to loss of activity by chymotrypsin digestion has been demonstrated previously (Amir, Barker, Butt & Crooke,
1966).
A purified porcine FSH preparation (GLM-1-18 PG) with a potency of 13-5 NIHFSH-P1 was provided by Dr Maryce M. Jacobs of the University of Texas. This preparation was similar to that described previously (Jacobs & Ward, 1976) except that the sialic acid content was three times lower. All hormones were diluted in 0-9% NaCl solution and were stored frozen in small portions until immediately before use. The cells were incubated in the absence or presence of the hormones in Eagle's medium containing Earle's salts (pH 7-4; Grand Island Biological Co.), 25 mM-Ar-2-hydroxyethylpiperazine-7V'-2-ethanesulphonic acid (HEPES) buffer (Calbiochem), 2-2 g NaHC03/l (Grand Island Biological Co.) and 1% BSA fraction V. This is designated as Eagle's medium plus 1% BSA. Both [3H]cyclic AMP (specific activity 24 Ci/mmol) and unlabelled cyclic AMP were obtained from Schwarz Bio-Research Inc. (Division of Dickinson & Co., Orangeburg, New York 10962, U.S.A.).
Incubation ofgranulosa cells Portions of granulosa cells from small, medium and large follicles suspended in Eagle's medium plus 1% BSA were added to glass scintillation vials containing the hormone; cells from small and medium-sized follicles were dispensed in aliquots of 2 IO7 cells and cells from large follicles in aliquots of 5 IO6 cells. The final incubation volume was 1-0 ml. Between three and five replicate incubations of cells were used for each variable in each experiment. The incubations were carried out as described previously (Channing & LedwitzRigby, 1975) for 1-60 min under an atmosphere of 5% C02 in air at 37 °C in a shaking water bath. The reaction was stopped by placing the vials immediately in ice. The content of each vial was then transferred to 12 75 mm test-tubes kept on ice and the cells were separated from the incubation medium by centrifugation at 4 °C at 1000 ¿» for 5 min. The supernatant fraction was decanted and frozen until required for assay of the cyclic AMP released into the incubation medium. Cyclic AMP was extracted from the cells as described previously (Kolena & Channing, 1972; Ledwitz-Rigby, Gay, Stetson, & Young, 1977).
Assay of cyclic AMP The amount of cyclic AMP in the cell extract and the amount of cyclic AMP released into the incubation medium were determined by the competitive protein-binding assay described by Gilman (1970) with the modifications of Kolena & Channing (1972) and Mashiter, Mashiter, Hauger & Field (1973). Fifty microlitre portions of cell extract and 100 µ untreated incubation medium were assayed in duplicate for their cyclic AMP content. The results are expressed as pmol cyclic AMP/5 107 cells. Using 1-25 pmol cyclic AMP as a standard after every ten unknown samples, the intra-assay coefficient of variation was less than 16% and for 30 randomly selected assays the between assay coefficient of variation was less than 15%. Statistical comparisons were made by Student's Mest. RESULTS
Effects of ovine, porcine and human FSH on intracellular accumulation of cyclic AMP After incubation for 15 min, 0-1 µg human FSH was effective in stimulating the intracellular accumulation of cyclic AMP in granulosa cells from small (Fig. lc;n= 15, < 0-001) and
o Q.
CU
.
U 50
10
(15)
100 1 10 01 001 Hormone concentration ^g/ml) Fig. 2. Effect of incubating porcine granulosa cells from medium follicles for 15 min with (a) ovine FSH pre-incubated with antiserum to LH, (b) porcine FSH or (c) human FSH on the intracellular accumulation of cyclic AMP. Results are means ± s.e.m. ; numbers of observations are given in 0
parentheses.
medium (Fig. 2c; n= 15, P 0-05). Some stimulation was observed after incubation of 1 µg (128 + 9 (s.e.m.) %; «=10, P
AND OF OVINE AND HUMAN LUTEINIZING HORMONE ON THE ACCUMULATION OF CYCLIC AMP BY PORCINE GRANULOSA CELLS CORNELIA P. CHANNING Department of Physiology, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, Maryland21201, U.S.A. ADA M. LINDSEY
AND
(Received 11 January 1978) SUMMARY
The effects of ovine, porcine and human FSH, and ovine and human LH on the accumulation of cyclic AMP by porcine granulosa cells obtained from follicles at various stages of maturation were investigated. During incubation periods of 15 min, 10 \g=m\g ovine FSH pretreated with antiserum to LH or 10 \g=m\ghuman FSH resulted in an 11- to 18-fold, five\x=req-\ to ninefold, and less than a twofold increase in intracellular accumulation of cyclic AMP by granulosa cells from small (1\p=n-\2 mm), medium (3\p=n-\5 mm) and large (6\p=n-\12 mm) follicles respectively. Similar patterns of response occurred with addition of porcine FSH. After incubation for 30 and 60 min with ovine, porcine or human FSH, significant accumulation of cyclic AMP in the incubation medium occurred with cells obtained from small and medium-sized follicles. After 60 min of incubation with FSH the accumulation of cyclic AMP in the incubation medium exceeded the intracellular cyclic AMP levels in granulosa cells from small and medium-sized follicles. During incubation periods of 15 min, 1\m=.\0\g=m\govine LH resulted in less than a twofold, a fourfold and greater than a tenfold increase in intracellular accumulation of cyclic AMP by granulosa cells from small, medium and large follicles respectively. Addition of human LH brought about a similar response. Incubation periods of 30 and 60 min with 1\m=.\0 \g=m\g ovine or human LH resulted in significant accumulation of cyclic AMP in the incubation medium by granulosa cells from large follicles; cyclic AMP content in the incubation medium was greater after 60 min compared with 30 min of incubation. It was concluded that ovine FSH pretreated with an antiserum to LH had similar effects on cyclic AMP levels as did purified human and porcine FSH, and that the stimulatory effects of the less pure ovine FSH were probably not due to an impurity in the FSH preparation. Porcine granulosa cells obtained from small follicles should be suitable as an in-vitro FSH bioassay while granulosa cells obtained from large follicles should be suitable as an in-vitro LH bioassay. INTRODUCTION
Gonadotrophin-mediated ovarian responses depend on not only the concentration of biologically active hormone but also the responsiveness of the target cell. It is generally believed that the first step in the mechanism of action of these glycoprotein hormones is their interaction with specific receptors in the target cells, which subsequently stimulates the formation of cyclic AMP from ATP (Robinson, Butcher & Sutherland, 1971). The cyclic AMP thus generated leads to biological effects on cellular metabolism and steroido¬ genesis and has been designated the second messenger of hormone action.
previously that ovine follicle-stimulating hormone (FSH) against luteinizing hormone (LH) has a greater stimulatory effect on the accumulation of cyclic AMP in cells obtained from small and medium follicles than in cells from large follicles (Lindsey & Channing, 1976). Since the ovine FSH pre¬ paration was not highly purified, the possibility existed that an impurity therein might be responsible for its action. To rule out this possibility, the effect of the less pure ovine FSH was compared with the effect of more highly purified FSH preparations from porcine and human sources. The effect of LH on granulosa cells in previous studies had been examined by measuring only the accumulation of cyclic AMP within the cells. In the present studies, measurement of the accumulation of cyclic AMP within the cells and in the incubation medium was used to examine the stimulatory effects of the gonadotrophins on porcine granulosa cells from small, medium and large follicles. The experiments were also designed to determine whether there were any differences in the response of porcine granulosa cells to gonadotrophins obtained from porcine, human and ovine sources. It has been demonstrated pretreated with an antiserum
MATERIALS AND METHODS
Granulosa cells Porcine ovaries were obtained within 15 min of death at a local meat-packing factory and were transported to the laboratory in ice-cold 0-9% NaCl solution containing 100 units penicillin and 100 µg streptomycin/ml. Granulosa cells were obtained from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles as described by Channing & Ledwitz-Rigby (1975). Granulosa cells were separated from follicular fluid by centrifugation at 600 g for 5 min and were washed with Eagle's medium three times. After resuspension in Eagle's medium (pH 7-4; Grand Island Biological Co., Grand Island, New York, U.S.A.) plus 1% bovine serum albumin (BSA) fraction V (Sigma Chemical Company), the granulosa cells were kept overnight at 4 °C until 1 h before use when they were allowed to equilibrate with room temperature. With dye exclusion as an indication of cell viability, the cells were counted in a haemocytometer in 0-06% trypan blue. Hormones and chemicals 1-10 i.u. NIH-oFSH-Sl/mg) and ovine LH
Ovine FSH (NIH-oFSH-SlO; (NIH-0LH-SI8; 1-03 i.u. NIH-oLH-Sl/mg) were used. The ovine FSH preparation contained less than 0-01 i.u. NIH-LH-Sl/mg and the ovine LH less than 0-05 i.u. NIH-FSH-Sl/mg. The potencies of the FSH and LH preparations were determined by the Steelman-Pohley bioassay (Steelman & Pohley, 1953) and the ovarian ascorbic acid depletion assay (Parlow, 1961) respectively. These hormones were supplied by the Hormone Distribution Officer of the National Institute of Arthritis and Metabolic and Digestive Diseases, NIH. Antiserum to bovine LH, generated in a mare (Snook, 1968), was donated by Dr William Hansel of Cornell University, and was used to neutralize the LH contaminant in the ovine FSH preparation by the following procedure. Various concentrations of ovine FSH and LH were pre-incubated with the antiserum to LH for 1 h at 37 °C before the granulosa cells were added. Antiserum dilutions 1 : 500 to 1 : 5000 were used depending on the concen¬ tration of LH to be neutralized. The dilutions of antiserum were determined experimentally; estimates of LH contaminating the ovine FSH preparation were calculated and ovine LH was pre-incubated with the antiserum to LH to demonstrate that the antiserum was effective at neutralizing the stimulatory effect of LH on granulosa cell accumulation of cyclic AMP. We have shown previously (Lindsey & Channing, 1976, 1978) that pre-incubation of ovine FSH with antiserum to LH can neutralize the ability of contaminating LH to stimulate the release of cyclic AMP in porcine granulosa cells. Therefore, in all the present experiments, ovine FSH was pretreated with antiserum to LH.
Highly purified human LH (LER-1705, potency 3800 i.u./mg and FSH activity 3 i.u./mg) and human FSH (LER-1577c, potency 880 i.u./mg) were provided by Dr L. E. Reichert, Jr. The potencies of human FSH and LH are expressed in terms of the Second International Reference Preparation of Human Menopausal Gonadotrophin Standard (2nd IRP-hMG standard). The FSH preparation had been pretreated with chymotrypsin to inactivate selectively the contaminating LH (Reichert, 1967); the residual LH activity in the FSH preparation had a potency of 5-7 i.u./mg. The resistance of FSH to loss of activity by chymotrypsin digestion has been demonstrated previously (Amir, Barker, Butt & Crooke,
1966).
A purified porcine FSH preparation (GLM-1-18 PG) with a potency of 13-5 NIHFSH-P1 was provided by Dr Maryce M. Jacobs of the University of Texas. This preparation was similar to that described previously (Jacobs & Ward, 1976) except that the sialic acid content was three times lower. All hormones were diluted in 0-9% NaCl solution and were stored frozen in small portions until immediately before use. The cells were incubated in the absence or presence of the hormones in Eagle's medium containing Earle's salts (pH 7-4; Grand Island Biological Co.), 25 mM-Ar-2-hydroxyethylpiperazine-7V'-2-ethanesulphonic acid (HEPES) buffer (Calbiochem), 2-2 g NaHC03/l (Grand Island Biological Co.) and 1% BSA fraction V. This is designated as Eagle's medium plus 1% BSA. Both [3H]cyclic AMP (specific activity 24 Ci/mmol) and unlabelled cyclic AMP were obtained from Schwarz Bio-Research Inc. (Division of Dickinson & Co., Orangeburg, New York 10962, U.S.A.).
Incubation ofgranulosa cells Portions of granulosa cells from small, medium and large follicles suspended in Eagle's medium plus 1% BSA were added to glass scintillation vials containing the hormone; cells from small and medium-sized follicles were dispensed in aliquots of 2 IO7 cells and cells from large follicles in aliquots of 5 IO6 cells. The final incubation volume was 1-0 ml. Between three and five replicate incubations of cells were used for each variable in each experiment. The incubations were carried out as described previously (Channing & LedwitzRigby, 1975) for 1-60 min under an atmosphere of 5% C02 in air at 37 °C in a shaking water bath. The reaction was stopped by placing the vials immediately in ice. The content of each vial was then transferred to 12 75 mm test-tubes kept on ice and the cells were separated from the incubation medium by centrifugation at 4 °C at 1000 ¿» for 5 min. The supernatant fraction was decanted and frozen until required for assay of the cyclic AMP released into the incubation medium. Cyclic AMP was extracted from the cells as described previously (Kolena & Channing, 1972; Ledwitz-Rigby, Gay, Stetson, & Young, 1977).
Assay of cyclic AMP The amount of cyclic AMP in the cell extract and the amount of cyclic AMP released into the incubation medium were determined by the competitive protein-binding assay described by Gilman (1970) with the modifications of Kolena & Channing (1972) and Mashiter, Mashiter, Hauger & Field (1973). Fifty microlitre portions of cell extract and 100 µ untreated incubation medium were assayed in duplicate for their cyclic AMP content. The results are expressed as pmol cyclic AMP/5 107 cells. Using 1-25 pmol cyclic AMP as a standard after every ten unknown samples, the intra-assay coefficient of variation was less than 16% and for 30 randomly selected assays the between assay coefficient of variation was less than 15%. Statistical comparisons were made by Student's Mest. RESULTS
Effects of ovine, porcine and human FSH on intracellular accumulation of cyclic AMP After incubation for 15 min, 0-1 µg human FSH was effective in stimulating the intracellular accumulation of cyclic AMP in granulosa cells from small (Fig. lc;n= 15, < 0-001) and
o Q.
CU
.
U 50
10
(15)
100 1 10 01 001 Hormone concentration ^g/ml) Fig. 2. Effect of incubating porcine granulosa cells from medium follicles for 15 min with (a) ovine FSH pre-incubated with antiserum to LH, (b) porcine FSH or (c) human FSH on the intracellular accumulation of cyclic AMP. Results are means ± s.e.m. ; numbers of observations are given in 0
parentheses.
medium (Fig. 2c; n= 15, P 0-05). Some stimulation was observed after incubation of 1 µg (128 + 9 (s.e.m.) %; «=10, P
