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The effect of luteinizing hormone and adenosine 3′,5′-monophosphate on progesterone biosynthesis by bovine granulosa cells in culture
PRELIMINARY
NOTES
THE EFFECT OF LUTEINIZING HORMONE AND ADENOSINE 3’,5’MONOPHOSPHATE ON PROGESTERONE BIOSYNTHESIS BY BOVINE GRANULOSA CELLS IN CULTURE V. J. CIRILLO, 0. F. ANDERSEN, E. A. HAM and R. B. L. GWATKIN, Merck Institute for Therapeutic Research, Rahway, NJ. 07065, USA
The isolation of specific endocrine cell types and study of their biosynthetic capabilities under well-defined conditions has contributed much to the understanding of hormone production and control. This procedure has been used recently to investigate progesterone synthesis by ovarian cells. With incubations of a few hours duration Bjersing & Carstensen [l] demonstrated the conversion of exogenous pregnenolone to progesterone by porcine ovarian granulosa cells, and Ryan & Petro [7] found human granulosa cells to be much more active than thecal cells in this conversion. Channing, using tissue culture, showed de novo progesterone biosynthesis by equine [2] and human [3] ovarian granulosa cells. Schomberg ES]has reported progesterone production by bovine granulosa cells in culture production by bovine granulosa cells in culture. This note describes the biosynthesis of progesterone by tissue cultures of granulosa cells from bovine ovarian follicles, and the stimulation of this synthesis by luteinizing hormone (LH) and adenosine 3’,5’-monophosphate (3’,5’-cyclic AMP).
Tissue culture methodology Follicles were dissected aseptically from ovaries freshly collected at the abbatoir, and the interior walls scraped to discharge the loosely attached granulosa cells into the tissue culture medium. This consisted of chemically defined
medium 199 ]4] supplemented with 10 % fetal calf serum. After a low-speed centrifugation the cells were resuspended in fresh medium, and lo6 cells in 5 ml of medium were added to each of a series of Falcon 25 cm2 culture flasks. The culture medium was replaced the next day, and this was followed by routine medium changes every 2 or 3 days. By the second day of incubation at 37°C groups of pleomorphic cells, containing prominent granules, were seen attached to the plastic surface. By the sixth day these cells had formed a complete monolayer (fig. 1). Cultures could be maintained for only about two weeks. Attempts to establish new cultures from existing ones by transferring cells with trypsin or pronase were unsuccessful. The number of cells in a culture was determined by removing the medium and adding 1 ml of 0.1% crystal violet in 0.1 M citric acid to each flask. After 15 min the stained nuclei were counted with a hemocytometer.
Progesterone analysis Progesterone biosynthesis was studied initially by extracting the medium and cells separately with redistilled ethyl acetate. Later the medium alone was extracted. l*C-4Progesterone was added prior to the extraction to estimate recoveries, but was omitted from later experiments since recoveries were always 99-100 %. The extracts were reduced to dryness under a stream of nitrogen, dissolved in redistilled carbon disulfide and subjected to gas-liquid chromatography. A Barber Colman model 5000 gas chromatograph was used employing a flame detector, and a 6 ft x 4 mm glass U-tube containing silanized 100-200 mesh Gas-Chrom P coated with 1.7 y0 SE-30. The column temperature was 230°C and the argon inlet pressure 30 psi. Exptl Cell Res 57
140
V. J. Cirillo
et al.
Fig. I. Bovine granulosa cells in culture. On the left is a culture on the 2nd day of incubation. Small refractile cells are mostly erythrocytes. On the right is a culture on the 6th day when a complete monolayer of granuloss cells has formed. Phase-contrast, x 130.
The identity of the progesterone peak was further established by examining the extracts on an LKB model 9000 gas chromatographmass spectrometer. The peak in the gas chromatograph was found to possess m/e values for the molecular ion and fragmentation products in agreement with an authentic specimen of progesterone. Experiments
and Results
The rate of progesterone synthesis over a fiveday period is shown in fig. 2. It is apparent that the cells actively synthesized progesterone during the entire period, secreting it into the medium with little accumulation within the cells themselves. Progesterone secretion varied little between replicate cultures prepared from the same pool of cells, but there was considerable variability when cultures were prepared from ovaries collected at different times (tabIe 1). This variation could not be correlated with any particular stage Exptl
Cell Res 57
of the estrus cycle, in so far as this could be established by the appearance of the follicles and corpora lutea. To determine whether cultures had retained responsiveness to gonadotrophin, bovine LH (10 pug/ml medium, a concentration equivalent to 2 pg NIH-LH-B6/ml in an assay based on the stimulation of cyclic AMP levels in slices of bovine corpus luteum) was added to the cultures at various times ranging from 1 to 6 days of cultivation and was included in subsequent medium changes. The progesterone output of the cultures was determined at various times, ranging from 1 to 4 days after the LH addition. Replicate cultures, prepared from the same pool of granulosa cells, served as controls or received 3’,5’-cyclic AMP (1O-3 M) plus equimolar theophylline continuously from day 5 of incubation. Further study indicated that theophylline, an inhibitor of the phosphodiesterase which degrades 3’S’-cyclic AMP, was an unnecessary
Progesterone biosynthesis in bovine granulosa cell cultures addition. Presumably, this is due to minimal phosphodiesterase activity in the cultures. Table 2 summarizes the results obtained. The addition of LH increased the progesterone levels in the medium in 28 Y! of the cultures. The incidence was slightly higher on a per cell basis. Response to 3’,5’-cyclic AMP was more consistent, occurring in 64% of the cultures on a per ml basis and in 100 % of cultures on a per cell basis. A possible reason for the discrepancy in these measurements may be the reduction in cell numbers per culture produced by LH and 3’,5’-cyclic AMP (see footnote to table 2) which would tend to decrease the progesterone output per ml of medium and increase the apparent output per cell. A similar toxicity of 3’,5’-cyclic AMP for HeLa and L cells was observed by Ryan & Heidrick [6]. As with variation in unstimulated progesterone output, there was no apparent correlation between responsiveness to LH and stage of the estrus cycle, judged by inspection of the ovaries. To establish whether continuous stimulation by gonadotrophin in vitro is required for progesterone synthesis, the fetal calf serum, which may have contained
141
Table 1. Progesterone synthesis by granulosa
cell cultures (variation within and between cell pools)
Progesterone &z/ml/2 days) Final cell concentration ( x 10-6/ml) Progesterone WceW days)
Single pool of cells”
Series of different cell poolsb
0.53 rt 0.04
0.86kO.59
0.23 + 0.02
0.64 k 0.31
2.20 + 0.20
1.42 k 0.88
’ From 12 ovaries. Data are means of quadruplicate cultures + S.D. Progesterone synthesis measured between 5th and 7th day. b Thirty-three different pools. Data are means + S.D. Progesterone synthesis measured beween 7th and 9th day.
gonadotrophins, was replaced by serum from hypophysectomized rats. Unfortunately, the rat serum was toxic. However, replacement of the fetal calf serum on the 5th day of cultivation with 0.1 Y0 bovine plasma albumin did not interrupt the synthesis of progesterone by the cells over the next 48 h, suggesting that the continual presence of LH is not needed for progesterone production. Table 2. Effect of LH and 3’,5’-Cyclic AMP
on progesterone synthesis by bovine granulosa cells
j &---+--~----? LH
3’,5’-Cyclic AMP
0
2
3
4
5
Abscissa: Days in culture; ordinate: picograms progesterone/cell/day. Fig. 2. Biosynthesis of progesterone by bovine granulosa ceils in culture. Solid line represents progesterone in medium; broken line that in cells. Medium changes were made on the first, second and third days of cultivation so that rates of synthesis were calculated from the amount of progesterone which accumulated between l-2, 2-3 and 3-5 days in culture.
Per ml Per cell
13/45 (28 %) 2.3 (1.3-5.8) 13/38 (34 %) 1.7 (1.3-3.8)
Per ml Per cell
7/11 (64%) 1.7 (1.34.0) 6/6 (100 %) 2.4 (1.6-4.7)
a Starting on 1st to 6th day of cultivation. b Progesterone output per ml medium or per cell determined for a 2-day period beginning l-4 days after treatment. c An increase 25 % greater than controls was scored as a stimulation. In 10 out of 13 experiments LH reduced cell counts by 15-59 %. In 5 of 6 experiments 3’,5’-cyclic AMP reduced cell counts by 17-70 %. a Mean + range. Exptl
Cell Res 57
142
F. Wunderlich and D. Peyk
Discussion These experiments have demonstrated that bovine granulosa cell cultures are capable of synthesizing progesterone and that in some cultures this biosynthesis is responsive to LH. However, the conditions for such a response remain unknown and at present are uncontrollable. An apparent lack of correlation with the estrus cycle could suggest that local conditions within the tissue are more important than the overall condition of the animal. Alternatively, the unresponsiveness of many cultures to LH may result from an erratic loss of functional activity under in vitro conditions. If so, responsiveness to 3’,5’cyclic AMP, a common mediator for several trophic hormones [5], may represent a more general mechanism which tends to be retained even under in vitro conditions. Retention of response to 3’,5’-cyclic AMP, with simultaneous loss of responsiveness to trophic hormones, was noted in steroid biosynthesis by monolayers of mouse testicular interstitial cells [9]. We are grateful to Mr E. L. Rickes for the generous gift of bovine LH (M632-25A).
REFERENCES 1. Bjersing, L & Carstensen, H, J reprod fertil 14 (1967) 101. 2. Channing, C P, Nature 210 (1966) 1266. 3. Charming, C P, Butt, W R & Crooke, A C, 3rd Intern congr endocrinol, Mexico, June 1968, Intern congr series, no. 157. Abstr 306, p. 123. Excerpta Medica Found, New York (1968). 4. Morgan, J F, Morton, H J & Parker, R C, Proc sot exptl biol med 73 (19.50) 1. 5. Robison, G A, Butcher, R W & Sutherland, E W, Ann rev biochem 37 (1968) 149. 6. Ryan, W L & Heidrick, M L, Science 162 (1968) 1484. Ryan, K J & Petro, Z, J clin endocr 26 (1966) 46. s7- Schomberg, D W, J endocr 38 (1967) 359. 9: Shin, S, Endocrinology 81 (1967) 440. Received March 25, 1969
Exptl Cell Res 57
ANTIMITOTIC AGENTS AND MACRONUCLEAR DIVISION OF CILIATES II. Endogenous Recovery from Colchicine and Colcemid-a new Method of Synchronization in Tetrahymena pyriformis GL F. WUNDERLICH and D. PEYK, Division of Cell Biology, Institute of Biology II, University of Freiburg i. Br., Germany
A good deal of the present work in cell biology is concerned with the problems of intracellular regulation processes. Of particular advance in studying these problems are methods of synchronizing cells with respect to their cell cycle. Synchronization methods are of special importance in a great many investigations using the ciliate protozoan Tetrahymena pyriformis, one of the most favourable organisms in cell research [20, 251. The first method to synchronize populations of this organism has been developed by Scherbaum & Zeuthen who treated the cells with a series of temperature shifts [13, 261. Other synchronizing methods for this organism followed (e.g. [8, 10, 15, 161). One of the various procedures to achieve cell cycle synchronization in eumitotic cells involves the metaphase arresting alkaloids colchicine and Colcemid. By reversal of the inhibition of mitosis-in connection with the method of Terasima & Tolmach [19]-one can attain a mitotic index of 80-95 % [9, 181. The antimitotic agents colchicine and Colcemid are thought of as acting on the protein subunits of the spindle microtubules. Since the occurrence of microtubular structures has been reported recently for the macronucleus of T. pyriformis amicronucleate strain GL [l, 4, 211, it was tempting to test whether antimitotic agents could also bring about a synchronization in T. pyriformis, the division of which is generally referred to as “amitotic”, which is thought of as a non-ordered pinching into daughter nuclei. In a previous paper [24] it has been de-
NOTES
THE EFFECT OF LUTEINIZING HORMONE AND ADENOSINE 3’,5’MONOPHOSPHATE ON PROGESTERONE BIOSYNTHESIS BY BOVINE GRANULOSA CELLS IN CULTURE V. J. CIRILLO, 0. F. ANDERSEN, E. A. HAM and R. B. L. GWATKIN, Merck Institute for Therapeutic Research, Rahway, NJ. 07065, USA
The isolation of specific endocrine cell types and study of their biosynthetic capabilities under well-defined conditions has contributed much to the understanding of hormone production and control. This procedure has been used recently to investigate progesterone synthesis by ovarian cells. With incubations of a few hours duration Bjersing & Carstensen [l] demonstrated the conversion of exogenous pregnenolone to progesterone by porcine ovarian granulosa cells, and Ryan & Petro [7] found human granulosa cells to be much more active than thecal cells in this conversion. Channing, using tissue culture, showed de novo progesterone biosynthesis by equine [2] and human [3] ovarian granulosa cells. Schomberg ES]has reported progesterone production by bovine granulosa cells in culture production by bovine granulosa cells in culture. This note describes the biosynthesis of progesterone by tissue cultures of granulosa cells from bovine ovarian follicles, and the stimulation of this synthesis by luteinizing hormone (LH) and adenosine 3’,5’-monophosphate (3’,5’-cyclic AMP).
Tissue culture methodology Follicles were dissected aseptically from ovaries freshly collected at the abbatoir, and the interior walls scraped to discharge the loosely attached granulosa cells into the tissue culture medium. This consisted of chemically defined
medium 199 ]4] supplemented with 10 % fetal calf serum. After a low-speed centrifugation the cells were resuspended in fresh medium, and lo6 cells in 5 ml of medium were added to each of a series of Falcon 25 cm2 culture flasks. The culture medium was replaced the next day, and this was followed by routine medium changes every 2 or 3 days. By the second day of incubation at 37°C groups of pleomorphic cells, containing prominent granules, were seen attached to the plastic surface. By the sixth day these cells had formed a complete monolayer (fig. 1). Cultures could be maintained for only about two weeks. Attempts to establish new cultures from existing ones by transferring cells with trypsin or pronase were unsuccessful. The number of cells in a culture was determined by removing the medium and adding 1 ml of 0.1% crystal violet in 0.1 M citric acid to each flask. After 15 min the stained nuclei were counted with a hemocytometer.
Progesterone analysis Progesterone biosynthesis was studied initially by extracting the medium and cells separately with redistilled ethyl acetate. Later the medium alone was extracted. l*C-4Progesterone was added prior to the extraction to estimate recoveries, but was omitted from later experiments since recoveries were always 99-100 %. The extracts were reduced to dryness under a stream of nitrogen, dissolved in redistilled carbon disulfide and subjected to gas-liquid chromatography. A Barber Colman model 5000 gas chromatograph was used employing a flame detector, and a 6 ft x 4 mm glass U-tube containing silanized 100-200 mesh Gas-Chrom P coated with 1.7 y0 SE-30. The column temperature was 230°C and the argon inlet pressure 30 psi. Exptl Cell Res 57
140
V. J. Cirillo
et al.
Fig. I. Bovine granulosa cells in culture. On the left is a culture on the 2nd day of incubation. Small refractile cells are mostly erythrocytes. On the right is a culture on the 6th day when a complete monolayer of granuloss cells has formed. Phase-contrast, x 130.
The identity of the progesterone peak was further established by examining the extracts on an LKB model 9000 gas chromatographmass spectrometer. The peak in the gas chromatograph was found to possess m/e values for the molecular ion and fragmentation products in agreement with an authentic specimen of progesterone. Experiments
and Results
The rate of progesterone synthesis over a fiveday period is shown in fig. 2. It is apparent that the cells actively synthesized progesterone during the entire period, secreting it into the medium with little accumulation within the cells themselves. Progesterone secretion varied little between replicate cultures prepared from the same pool of cells, but there was considerable variability when cultures were prepared from ovaries collected at different times (tabIe 1). This variation could not be correlated with any particular stage Exptl
Cell Res 57
of the estrus cycle, in so far as this could be established by the appearance of the follicles and corpora lutea. To determine whether cultures had retained responsiveness to gonadotrophin, bovine LH (10 pug/ml medium, a concentration equivalent to 2 pg NIH-LH-B6/ml in an assay based on the stimulation of cyclic AMP levels in slices of bovine corpus luteum) was added to the cultures at various times ranging from 1 to 6 days of cultivation and was included in subsequent medium changes. The progesterone output of the cultures was determined at various times, ranging from 1 to 4 days after the LH addition. Replicate cultures, prepared from the same pool of granulosa cells, served as controls or received 3’,5’-cyclic AMP (1O-3 M) plus equimolar theophylline continuously from day 5 of incubation. Further study indicated that theophylline, an inhibitor of the phosphodiesterase which degrades 3’S’-cyclic AMP, was an unnecessary
Progesterone biosynthesis in bovine granulosa cell cultures addition. Presumably, this is due to minimal phosphodiesterase activity in the cultures. Table 2 summarizes the results obtained. The addition of LH increased the progesterone levels in the medium in 28 Y! of the cultures. The incidence was slightly higher on a per cell basis. Response to 3’,5’-cyclic AMP was more consistent, occurring in 64% of the cultures on a per ml basis and in 100 % of cultures on a per cell basis. A possible reason for the discrepancy in these measurements may be the reduction in cell numbers per culture produced by LH and 3’,5’-cyclic AMP (see footnote to table 2) which would tend to decrease the progesterone output per ml of medium and increase the apparent output per cell. A similar toxicity of 3’,5’-cyclic AMP for HeLa and L cells was observed by Ryan & Heidrick [6]. As with variation in unstimulated progesterone output, there was no apparent correlation between responsiveness to LH and stage of the estrus cycle, judged by inspection of the ovaries. To establish whether continuous stimulation by gonadotrophin in vitro is required for progesterone synthesis, the fetal calf serum, which may have contained
141
Table 1. Progesterone synthesis by granulosa
cell cultures (variation within and between cell pools)
Progesterone &z/ml/2 days) Final cell concentration ( x 10-6/ml) Progesterone WceW days)
Single pool of cells”
Series of different cell poolsb
0.53 rt 0.04
0.86kO.59
0.23 + 0.02
0.64 k 0.31
2.20 + 0.20
1.42 k 0.88
’ From 12 ovaries. Data are means of quadruplicate cultures + S.D. Progesterone synthesis measured between 5th and 7th day. b Thirty-three different pools. Data are means + S.D. Progesterone synthesis measured beween 7th and 9th day.
gonadotrophins, was replaced by serum from hypophysectomized rats. Unfortunately, the rat serum was toxic. However, replacement of the fetal calf serum on the 5th day of cultivation with 0.1 Y0 bovine plasma albumin did not interrupt the synthesis of progesterone by the cells over the next 48 h, suggesting that the continual presence of LH is not needed for progesterone production. Table 2. Effect of LH and 3’,5’-Cyclic AMP
on progesterone synthesis by bovine granulosa cells
j &---+--~----? LH
3’,5’-Cyclic AMP
0
2
3
4
5
Abscissa: Days in culture; ordinate: picograms progesterone/cell/day. Fig. 2. Biosynthesis of progesterone by bovine granulosa ceils in culture. Solid line represents progesterone in medium; broken line that in cells. Medium changes were made on the first, second and third days of cultivation so that rates of synthesis were calculated from the amount of progesterone which accumulated between l-2, 2-3 and 3-5 days in culture.
Per ml Per cell
13/45 (28 %) 2.3 (1.3-5.8) 13/38 (34 %) 1.7 (1.3-3.8)
Per ml Per cell
7/11 (64%) 1.7 (1.34.0) 6/6 (100 %) 2.4 (1.6-4.7)
a Starting on 1st to 6th day of cultivation. b Progesterone output per ml medium or per cell determined for a 2-day period beginning l-4 days after treatment. c An increase 25 % greater than controls was scored as a stimulation. In 10 out of 13 experiments LH reduced cell counts by 15-59 %. In 5 of 6 experiments 3’,5’-cyclic AMP reduced cell counts by 17-70 %. a Mean + range. Exptl
Cell Res 57
142
F. Wunderlich and D. Peyk
Discussion These experiments have demonstrated that bovine granulosa cell cultures are capable of synthesizing progesterone and that in some cultures this biosynthesis is responsive to LH. However, the conditions for such a response remain unknown and at present are uncontrollable. An apparent lack of correlation with the estrus cycle could suggest that local conditions within the tissue are more important than the overall condition of the animal. Alternatively, the unresponsiveness of many cultures to LH may result from an erratic loss of functional activity under in vitro conditions. If so, responsiveness to 3’,5’cyclic AMP, a common mediator for several trophic hormones [5], may represent a more general mechanism which tends to be retained even under in vitro conditions. Retention of response to 3’,5’-cyclic AMP, with simultaneous loss of responsiveness to trophic hormones, was noted in steroid biosynthesis by monolayers of mouse testicular interstitial cells [9]. We are grateful to Mr E. L. Rickes for the generous gift of bovine LH (M632-25A).
REFERENCES 1. Bjersing, L & Carstensen, H, J reprod fertil 14 (1967) 101. 2. Channing, C P, Nature 210 (1966) 1266. 3. Charming, C P, Butt, W R & Crooke, A C, 3rd Intern congr endocrinol, Mexico, June 1968, Intern congr series, no. 157. Abstr 306, p. 123. Excerpta Medica Found, New York (1968). 4. Morgan, J F, Morton, H J & Parker, R C, Proc sot exptl biol med 73 (19.50) 1. 5. Robison, G A, Butcher, R W & Sutherland, E W, Ann rev biochem 37 (1968) 149. 6. Ryan, W L & Heidrick, M L, Science 162 (1968) 1484. Ryan, K J & Petro, Z, J clin endocr 26 (1966) 46. s7- Schomberg, D W, J endocr 38 (1967) 359. 9: Shin, S, Endocrinology 81 (1967) 440. Received March 25, 1969
Exptl Cell Res 57
ANTIMITOTIC AGENTS AND MACRONUCLEAR DIVISION OF CILIATES II. Endogenous Recovery from Colchicine and Colcemid-a new Method of Synchronization in Tetrahymena pyriformis GL F. WUNDERLICH and D. PEYK, Division of Cell Biology, Institute of Biology II, University of Freiburg i. Br., Germany
A good deal of the present work in cell biology is concerned with the problems of intracellular regulation processes. Of particular advance in studying these problems are methods of synchronizing cells with respect to their cell cycle. Synchronization methods are of special importance in a great many investigations using the ciliate protozoan Tetrahymena pyriformis, one of the most favourable organisms in cell research [20, 251. The first method to synchronize populations of this organism has been developed by Scherbaum & Zeuthen who treated the cells with a series of temperature shifts [13, 261. Other synchronizing methods for this organism followed (e.g. [8, 10, 15, 161). One of the various procedures to achieve cell cycle synchronization in eumitotic cells involves the metaphase arresting alkaloids colchicine and Colcemid. By reversal of the inhibition of mitosis-in connection with the method of Terasima & Tolmach [19]-one can attain a mitotic index of 80-95 % [9, 181. The antimitotic agents colchicine and Colcemid are thought of as acting on the protein subunits of the spindle microtubules. Since the occurrence of microtubular structures has been reported recently for the macronucleus of T. pyriformis amicronucleate strain GL [l, 4, 211, it was tempting to test whether antimitotic agents could also bring about a synchronization in T. pyriformis, the division of which is generally referred to as “amitotic”, which is thought of as a non-ordered pinching into daughter nuclei. In a previous paper [24] it has been de-