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Induction by PGF2α of 20α-hydroxysteroid dehydrogenase in first generation corpora lutea of the rat
Molecular and Cellular Endocrinology
INDUCTION DEHYDROGENASE
of Physiology, Biodynamics,
Pub]. Comp.
BY PGF,, OF 20a-HYDROXYSTEROID IN FIRST GENERATION CORPORA LUTEA OF THE RAT
S. A. LAMPRECHT*, Department
3 (1975) 273-282. 0 North-Holland
H. V. HERLITZ
University
of Gdteborg,
Weizmann
Institute
Received 27 January 1975
and K. E. B. AHRBN
Giiteborg,
of Science,
Sweden
Rehovot,
and Department
qf’
Israel.
Accepted 15 April 1975
20a-Hydroxysteroid dehydrogenase (20a-OH-SDH) activity was determined in the first generation corpora lutea from prepubertal rats injected with 10 I.U. of pregnant mare’s serum gonadotrophin (PMSG) on day 30. The enzyme was not detectable in 1-9-day-old corpora lutea but a significant activity was seen on day 10. Enzyme activity increased during day 11 and day 12. In vivo administration of prostaglandin Fza (PGF& induced the enzyme in rats with corpora lutea older than 3 days. When prolactin was given concurrently with PGFZa, the corpus luteum activity of 20a-OH-SDH was lower than when PGFza was given alone. It is concluded that the present ‘corpus luteum model’ is suitable for further analysis of the cellular mechanisms of the luteolytic effect of prostaglandins (PGs) as well as of the role of gonadotrophins in the luteolytic process. Keywords:
20a-hydroxysteroid dehydrogenase; corpus luteum; PGF,,; prolactin.
pregnant mare’s serum gonadotrophin;
The luteal enzyme 20a-hydroxysteroid dehydrogenase (E.C. 1.1.1.149 ; 20a-OH-SDH) converts progesterone to the 20a-dihydroderivative which is essentially devoid of progestational activity in the rat (Wilcox and Wiest, 1960; Talwalker et al., 1966). The appearance of 20a-OH-SDH activity in corpora lutea previously devoid of the enzyme is an index of luteolysis (Wiest et al., 1968; Lamprecht et al., 1969; Wiest and Kidwell, 1969). In the rat ovary the activity of 20a-OH-SDH exhibits characteristic fluctuations during the oestrous cycle (Wiest, 1959; Pupkin et al., 1966; Bast and Melampy, 1972); it is barely detectable in the newly formed corpora lutea of pregnancy and pseudopregnancy (Lamprecht et al. 1969; Turolla et al., 1970; Strauss and Stambaugh, 1974). *In partial fulfilment of the requirements Weizmann Institute of Science.
for Ph. D. degree of the Graduate
School of the
214
S. A. Lamprecht et al.
The mechanisms
governing
modulation
of 20a-OH-SDH
activity
are not
fully understood, but there is strong evidence that prolactin suppresses the synthesis of the enzyme in the corpus luteum: pharmacological blockade of pituitary prolactin secretion results in induction of 20a-OH-SDH activity in corpora lutea of pregnant or pseudopregnant rats and the induction is prevented by administration of prolactin (Lamprecht et al., 1969; Hashimoto and Wiest, 1969b). Hashimoto and Wiest (1969a) have followed the appearance of the activity of 20a-OH-SDH in the first generation corpora lutea formed in prepubertal rat ovaries after synchronized ovulation triggered by exogenous pregnant mare’s serum gonadotrophin (PMSG). This technique in inducing formation of corpora lutea has the advantage that growth and development of the newly formed corpora lutea can be studied without the co-existence of corpora lutea in various stages of regression, as found in the ovary of adult cycling rats. Hashimoto and Wiest (1969a) injected 4 I.U. PMSG to Holzmann rats on day 26, leading to ovulation and corpus luteum formation early on day 29. The 20a-OH-SDH activity started to increase in these corpora lutea 4-5 days after the induced ovulation. In vivo administration of prostaglandin F,, (PGF*,) to pregnant and pseudopregnant rats results in premature termination of luteal function (Duncan and Pharriss, 1970; Pharriss et al., 1972) and it has recently been reported that PGF 2a can induce 20a-OH-SDH activity in corpora lutea of pregnant rats (Fuchs and Mok, 1974; Strauss and Stambaugh, 1974). There is evidence that this action of PGF,, is dependent on the degree of maturity of the corpus luteum (Gutknecht et al., 1969; Fuchs and Mok, 1974; Strauss and Stambaugh, 1974). The present study had the following .objectives: (i) to determine, by direct biochemical analysis of 20a-OH-SDH activity, the onset of spontaneous luteolysis in the first generation corpora lutea from PMSG-treated prepubertal rats, (ii) to examine the corpus luteum response to in vivo administration of PGF,, and (iii) to investigate whether prolactin suppresses a PGF,,-induced rise in 20a-OH-SDH activity in this type of corpora lutea.
MATERIALS
AND
METHODS
Animals and treatments Female Sprague-Dawley rats from Anticimex Ltd., Stockholm, Sweden were used. The animals were kept on a regular day and night schedule, and fed a standard diet and water ad libitum.
PGFza and 20a-OH-SDH
275
In order to obtain first generation corpora lutea, 10 I.U. PMSG (Gestyl, N.V. Organon, Oss, The Netherlands) were given subcutaneously to 30-day-old rats between 8 and 10 a.m. Ovulation occurred early in the morning of day 33 with formation of lo-20 corpora lutea per rat. Corpora lutea were excised with microscalpels with the aid of a dissecting microscope and kept in ice-cold 0.15 M NaCl before incubation; those isolated from 33-day-old rats, 6-10 h after ovulation, are designated as one-day-old corpora lutea with consecutive numbering up to 13-day-old corpora lutea derived from 45-day-old rats (fig. 1). Whole ovaries were trimmed of adherent fat and kept in cold saline before assay of 20a-OH-SDH. Three different sets of experiments were performed. In the first set, following administration of the ovulatory dose of PMSG, the rats did not receive any further treatment. Whole ovaries or newly formed corpora lutea were collected at different time intervals and assayed for 20a-OH-SDH activity. subcutaneously In the second set of experiments, PGF 2a was administered (250 ,ug, twice daily at 5 p.m. and 11 p.m.) to rats at various time intervals following the synchronized ovulation. PGFZa, supplied by the Upjohn Company of Canada, Don Mills, Ontario and ON0 Pharmaceutical Co., Ltd., Osaka, Japan, was dissolved in 9.5% ethanol to which was added a Na,CO,-water mixture (0.2 mg/ml) to achieve a final prostaglandin concentration of 0.5 mg/ml. Control rats received injections of 0.5 ml of vehicle. The rats were sacrificed 9 h after the last injection and the corpora lutea examined for 20a-OH-SDH. In a number of rats, PGFza (250 ug) was administered subcutaneously 6 days after the induced ovulation and the animals were divided into three groups: group I received PGFza 6 and 3 h prior to sacrifice, group II 12 and 6 h and group III 18 and 9 h before collection of corpora lutea. In the third set of experiments, 200 ug of ovine prolactin (National Institutes of Health, P-S-3 in 0.9 % saline) was injected concurrently with PGF,, (250 pg) to 39-day-old rats, six days after ovulation. Control rats received PGF,, only. The injections were given 18 and 9 h before the assay of luteal 20a-OH-SDH. The stated potency of the prolactin preparation was 15 I.U./mg; it was free of detectable luteinizing hormone (less than 0.0004 NIH-LH-SI units/mg) and follicle-stimulating hormone (less than 0.009 NIH-FSH-SI units/mg) activity. Reagents and substrates All chemicals and solvents were of analytical grade. 20a-Hydroxypregn4-en-3-one (Ikapharm, Ramat Gan, Israel) was examined by paper and gasliquid chromatography and found to be >98 % pure. NADP+ (Sigma Grade, Sigma Chemical Co., St. Louis, USA) was dissolved in the incubation medium immediately before use.
276
S. A. Lantprech~ et al.
The assay procedure followed closely that of Lamprecht et al. (1969). Tris buffer, 0.1 M (pH 8.0) containing 1 mm01 EDTA, 1 mmol cysteine and 10 mmoles nicotinamide per 1 was used as the medium for homogenization and incubation. The ovaries were removed, trimmed of adherent fat, and the corpora lutea excised. Each sample contained 10-20 corpora lutea derived from a single rat. The pooled corpora lutea were homogenized for 2 min when immersed in crushed ice and the homogenates were spun in a Sorvall refrigerated centrifuge at 15,000 g for 20 min. The supernatant was taken for protein determination according to Lowry et al. (1951). 20a-OH-SDH activity was assayed in 1 cm light path quartz cuvettes. Each cuvette was filled with 2.7 ml of the buffered medium, 1 pmole of NADP dissolved in 0.1 ml of the same medium, and 0.316 nmole of 20uhydroxypregn4-en-3-one dissolved in 0.1 ml ethanol. The cells were then preincubated at 37 “C for 10 min and the reaction was initiated by adding 0.1-0.3 ml of the tissue extract supernatant. NADPH generation was measured by continuously recording the absorbance at 340 nm. Activity of 20cc-OH-SDH was expressed in terms of milliunits (mU) of enzyme activity per mg 15,000 g supernatant fraction protein (mU/mg protein), where one unit of activity was defined as the formation of 1 pmole of NADPH per min at 37 “C under conditions of the assay,
RESULTS Administration of 10 I.U. of PMSG to 30-day-old prepubertal rats initiates the series of events shown in fig. 1. In another series of experiments (Hillensjb et al., 1974) it was shown that the PMSG-injected rats had an endogenous
corpus luteum -b
PM%
.
IOIU
I
I
I
I
I,
I
CL-12
CL-9
CL-6
I
,
I
n
I
I
I
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Rat age (days) Fig. 1. Schematic illustration of the PMSG model. CL-3, CL-6, CL-9 and CL-12 represent 3, 6, 9 and 12-day-old corpora lutea (CL), respectively.
277
PGFza and 20a-OH-SDH
Table 1. 20a-OH-SDH activity in whole ovaries and isolated corpora lutea of different ages from prepubertal rats injected with 10 I.U. PMSG on day 30. 20a-OH-SDH was measured in mU/mg protein, where mU = milliunits of enzyme activity, one unit being equal to the formation of 1 umole NADPH/min at 37 “C. Values are given as means & S.E.M. with the numbers of observations in parenthesis. N.D. = not detectable. Compartment analyzed
Rat age (days)
whole whole corpus corpus corpus corpus corpus
32 33 34-41 42 43 44 45
ovary ovary luteum luteum luteum luteum luteum
Corpus luteum age (days) before ovulation 1 (6-8 h) 2-9 10 11 12 13
20a-OH-SDH (mU/mg protein) N.D. N.D. N.D. 6 and 52 76f15 96 f 32 139 k 29
(2) (5) (4) (5)
release of LH in the afternoon of the 32nd day. The rats presented 12-1.5 ova in the tubal ampulla in the morning of day 33 corresponding to the normal number of ova found in the cycling rat. Whole ovaries collected 48 h after the administration of PMSG and corpora lutea dissected 6-9 h after ovulation were devoid of 20a-OH-SDH activity: l-9day-old corpora lutea also showed no measurable activity (table 1). A spontaneous rise in 20a-OH-SDH activity was observed in lo-day-old corpora lutea and enzyme activity increased progressively in older corpora lutea.
Table 2 Effect of PGFza on 20a-OH-SDH activity in the first generation corpora lutea of different ages from prepubertal rats injected with 10 I.U. PMSG on day 30. Abbreviations are as in table 1. PGFza (250 pg) was injected 18 and 9 h prior to isolation of corpora lutea. 20a-OH-SDH
(mU/mg protein)
Corpus luteum age (days) Control 2
3 4 6 7
N.D. N.D. N.D. N.D. N.D.
PGFz,-treated N.D. N.D. 6 i 0.9 17 + 2 34 i 5
(5) (5) (5)
278
S. A. Lampvecht
0
%a
et al.
plj5. Prolactin
Fig. 2. PGF,, (250 pg) and prolactin (200 pg) were injected concurrently S.C. to 39-day-old PMSG-treated rats, i.e. 6 days after ovulation. Two injections were given, 18 and 9 h prior to isolation of corpora lutea and determination of 20a-OH-SDH activity. There were 9 observations in one group (PGFza alone) and 8 in the other.
In vivo administration of PGF*, (250 pg/rat b.i.d.) resulted in the induction of 20a-OH-SDH activity in 4-day-old corpora lutea. Younger corpora lutea, l-3 days of age, were refractory to this action of PGFza (table 2). The effect of PGF,, was more pronounced in older corpora lutea: in 7-day-old corpora lutea the activity of 20a-OH-SDH was approximately 5-fold higher than in corpora lutea 3 days younger (table 2). The stimulation of 20a-OH-SDH in the corpora lutea was shown to be a rapid process. When PGF,, (250 pg) was given 6 and 3 h, 12 and 6 h, and 18 and 9 h, respectively, prior to isolation of 6-day-old corpora lutea, marked 20a-OH-SDH activity was observed as early as 6 h after the PGF,, injection. This stimulation did not become more pronounced after 12 and 18 h. In vivo administration of prolactin (KC. 200 pg/rat b.i.d.) partially prevented the PGF,,-induced rise of 20a-OH-SDH in the 7-day-old corpora lutea (fig. 2); enzyme activity was reduced by 50%, a difference which is statistically significant (P~0.05).
279
DISCUSSION In the present experiments, the first generation corpora futea had a life-span of lo-13 days, similar to that observed in the corpora lutea of the pseudopregnant rat. 20a-OH-SDH activity appeared spontaneously on day 10, and the enzyme activity increased during the following days. Hashimoto and Wiest (1969a) obtained a single generation of functional corpora lutea after administration of a lower.dose of PMSG (4 I.U.) to prepubertal rats at the age of 26 days. A spontaneous rise of ZOa-OH-SDH activity was found in corpora lutea 4-5 days ofd, at a time when no enzyme activity was found in corpora lutea of our PMSG model. The dose of PMSG used in relation to the normal puberty of the strain may be an important factor in causing this discrepancy. The luteolytic process in our experiments was triggered in 4-9-day-old corpora lutea by administration of PGF,,: activity of 20a-OH-SDH was observed as early as 6 h after administration of the drug. An effect of PGF2, in inducing luteal 20a-OH-SDH has also been observed in pregnant rats, both by determining the progestin pattern in the ovary (Behrmann et al., 1971) and by determination of 20a-OH-SDH activity in isolated corpora lutea and ovaries (Strauss and Stambaugh, 1974; Fuchs and Mok, 1974). The relatively rapid action of PGF,, in stimulating 20a-OH-SDH activity is in marked contrast to the 24-48 h Iatency of the effect of ergocornine, an alkaloid known to induce ZOa-OH-SDH activity in the corpora lutea of the pregnant and pseudopregnant rat (Lamprecht et al., 1969). The hormonal mechanisms involved in triggering the physiological luteolysis in the ovary of the rat are unclear. There is considerable evidence that the non-pregnant uterine endometrium produces a luteolysin in many mammals including the rat (e.g. Pharriss et al., 1972) and there is evidence in favour of prostaglandins as endogenous uterine luteolytic factors in some species (Blatchley and Donovan, 1969; McCracken, 1971). The results of the present study together with earlier investigations (e.g. Pharriss and Wyngarden, 1969; Behrman et al., 1971) show that exogenous PGFza mimics spontaneous luteolysis in the rat. However, factors other than PGFza also influence the onset of luteolysis. It has been suggested that prolactin and LH, the main components of the luteotrophic complex in the rat, may exert Iuteolytic actions, depending on the age and the endocrine status of the corpus luteum and the phase of the reproductive cycle (Rothchild, 1965; Malven, 1969). In our series of experiments, responsiveness to exogenous PGF,, in the first generation corpora lutea was clearly correlated with the age of the corpus luteua. Corpora lutea younger than 3 days did not respond to the action of the PGF2a, and the effect of PGFza in stimulating 20a-OH-SDH activity became morz pronounced
280
with increasing
S. A. Lamprecht
age. The magnitude
of the effect of PGF,,
has also been shown to depend on the age of corpora (Gutknecht et al., 1969; Fuchs and Mok, 1974).
in inducing
et al.
abortion
lutea in the pregnant
rat
An intriguing question is which mechanisms cause acquisition of responsiveness to exogenous PGF,, during a time-span of 24 h (between the 3rd and 4th day). It is possible that the endocrine environment to which the corpus luteum is exposed may play a crucial role in the timing of the acquisition of responsiveness to prostaglandins. It is therefore of interest to note that l-dayold corpora lutea already have receptor sites for LH and PGE, since they can respond to stimulation by either of these substances with an increase in CAMP (Herlitz et al., 1974). The view that prolactin maintains luteal function in the rat has been based, in the past, exclusively on results of biological assays, such as the support of pregnancy (Cutuly, 1941) and deciduomata formation (Evans et al., 1941) or the persistance of vaginal mucification in estrogen-treated, hypophysectomized rats (Astwood, 1941). It has been assumed that these effects reflect a stimulation of luteal progesterone synthesis. There is, however, increasing evidence that prolactin might owe its luteotrophic action mainly to an inhibitory effect on the synthesis of 20cr-OH-SDH and additional ovarian reductases (Lamprecht et al., 1969; Hashimoto and Wiest, 1969b; Zmigrod et al., 1972). The finding of the present study that concurrent administration of prolactin and PGFza resulted in an inhibitory influence on the induction of 20cr-OH-SDH in 7-day-old corpora lutea favours this hypothesis. A similar suppressive effect of prolactin on PGF,.-induced 20a-OH-SDH activity has recently been seen in corpora lutea of pregnancy (Strauss and Stambaugh, 1974). Establishment of pseudopregnancy, formation of deciduomata and direct determination of post-ovulatory secretion of progesterone have provided evidence for the functional capacity of the first set of corpora lutea in the study of Hashimoto and Wiest (1969a). We have not yet explored these parameters of luteal function in our experimental model. Herlitz et al. (1974) have, however, studied the in vitro effect of LH on the CAMP content of the same types of corpora lutea dissected l-7 days after ovulation in PMSG-treated rats. The young corpora lutea showed an extensive response while LLY produced only a minimal increase in CAMP in the old corpora lutea. The reason why aged corpora lutea responded less efficiently to stimulation by LH in vitro is unclear. An attractive hypothesis, although totally speculative, is that this behaviour reflects the initiation of the regression of corpus luteum. Some of the possibilities considered above concerning the development of are equally relevant to the explanation of responsiveness to exogenous PGF,, the dramatic quantitative change in response to LH as a function of corpus
PGF,,
luteum
281
and 20a-OH-SDH
age. These considerations
emphasize
the fact that the corpus luteum
in
its ephemeral existence is an ever-changing biochemical and structural entity, and that it is not meaningful to discuss how the corpus luteum behaves without considering its past history and present environment.
ACKNOWLEDGMENTS We want to thank Professor Hans Lindner for valuable suggestions. We are also indebted to the Endocrinology Study Section of NIH and N.V. Organon for the generous supply of hormones and Ono Pharmaceutical and Upjohn Companies for the gift of PGF,,. Skilled technical assistance was performed by Mrs Anita Sjbgren. The present investigation was supported by grants from the Swedish Medical Research Council (B74-03X-27), U.S. Public Health (2 ROl HD 0279.5), Ford Foundation, Population Council and from the Medical Faculty, University of Goteborg.
REFERENCES Astwood, E. B. (1941) Endocrinology 28, 309. Bast, J. D. and Melampy, R. M. (1972) Endocrinology 91, 1499. Behrman, H. R., Yoshinaga, K., Wyman, H. and Greep, R. 0. (1971) Am. J. Physiol. 221,189. Blatchley, F. R. and Donovan, B. T. (1969) Nature 221, 1065. Cutuly, E. (1941) Proc. Sot. Exptl. Biol. Med. 48, 315. Duncan, G. W. and Pharriss, B. B. (1970) Fed. Proc. 29, 1232. Evans, H. M., Simpson, M. E., Lyon, W. R. and Turpeinen, K. (1941) Endocrinology 28,933. Fuchs, A.-R. and Mok, E. (1974) Biol. Reprod. 10, 24. Gutknecht, G. D., Cornette, J. C. and Pharriss, B. B. (1969) Biol. Reprod. 1, 367. Hashimoto, I. and Wiest, W. G. (1969a) Endocrinology 84, 873. Hashimoto, I. and Wiest, W. G. (1969b) Endocrinology 84, 886. Herlitz, H., Hamberger, L., Rosberg, S. and Ah&n, K. (1974) Acta Endocrinol. 77, 737. Hillensjo, T., Barnea, A., Nilsson, L., Herlitz, H. and Ah&n, K. (1974) Endocrinology 95, 1762. Lamprecht, S. A., Lindner, H. R. and Strauss III, J. F. (1969) Biochim. Biophys. Acta 187,133. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193,265. Malven, P. V. (1969) In: The Gonads, Ed.: K. W. McKerns (Appleton-Century-Crofts, New YorIc) p. 367. McCracken, J. A. (1971) Ann. N.Y. Acad. Sci. 180, 465. Pharriss, B. B. and Wyngarden, L. J. (1969) Proc. Sot. Exptl. Biol. Med. 130, 92. Pharriss, B. B., Tillson, S. A. and Erickson, R. R. (1972) Rec. Prog. Horm. Res. 28, 51. Pupkin, M., Bratt, H., Weisz, J., Lloyd, C. W. and Balogh Jr, K. (1966) Endocrinology 79, 316. Rothchild, I. (1965) Acta Endocrinol. 49, 107. Strauss III, J. F. and Stambaugh, R. L. (1974) Prostaglandins 5, 73.
282
S. A. Lamprecht et al.
Talwalker, P. K., Krahenbuhl, C. and Desaulles, P. A. (1966) Nature 209, 86. Turolla, E., Baldratti, G. and Scrascia, E. (1970) Experientia 26, 418. Wiest, W. G. (1959) J. Biol. Chem. 234, 3115. Wiest, W. G., Kidwell, W. R. and Balogh Jr, K. (1968) Endocrinology 82, 844. Wiest, W. G. and Kidwell, W. R. (1969) In: The Gonads, Ed.: K. W. McKerns (AppletonCentury-Crofts, New York) p. 295. Wilcox, R. B. and Wiest, W. G. (1960) Endocrinology 67, 281. Zmigrod, A., Lindner, H. R. and Lamprecht, S. A. (1972) Acta Endocrinol. 69, 141.
INDUCTION DEHYDROGENASE
of Physiology, Biodynamics,
Pub]. Comp.
BY PGF,, OF 20a-HYDROXYSTEROID IN FIRST GENERATION CORPORA LUTEA OF THE RAT
S. A. LAMPRECHT*, Department
3 (1975) 273-282. 0 North-Holland
H. V. HERLITZ
University
of Gdteborg,
Weizmann
Institute
Received 27 January 1975
and K. E. B. AHRBN
Giiteborg,
of Science,
Sweden
Rehovot,
and Department
qf’
Israel.
Accepted 15 April 1975
20a-Hydroxysteroid dehydrogenase (20a-OH-SDH) activity was determined in the first generation corpora lutea from prepubertal rats injected with 10 I.U. of pregnant mare’s serum gonadotrophin (PMSG) on day 30. The enzyme was not detectable in 1-9-day-old corpora lutea but a significant activity was seen on day 10. Enzyme activity increased during day 11 and day 12. In vivo administration of prostaglandin Fza (PGF& induced the enzyme in rats with corpora lutea older than 3 days. When prolactin was given concurrently with PGFZa, the corpus luteum activity of 20a-OH-SDH was lower than when PGFza was given alone. It is concluded that the present ‘corpus luteum model’ is suitable for further analysis of the cellular mechanisms of the luteolytic effect of prostaglandins (PGs) as well as of the role of gonadotrophins in the luteolytic process. Keywords:
20a-hydroxysteroid dehydrogenase; corpus luteum; PGF,,; prolactin.
pregnant mare’s serum gonadotrophin;
The luteal enzyme 20a-hydroxysteroid dehydrogenase (E.C. 1.1.1.149 ; 20a-OH-SDH) converts progesterone to the 20a-dihydroderivative which is essentially devoid of progestational activity in the rat (Wilcox and Wiest, 1960; Talwalker et al., 1966). The appearance of 20a-OH-SDH activity in corpora lutea previously devoid of the enzyme is an index of luteolysis (Wiest et al., 1968; Lamprecht et al., 1969; Wiest and Kidwell, 1969). In the rat ovary the activity of 20a-OH-SDH exhibits characteristic fluctuations during the oestrous cycle (Wiest, 1959; Pupkin et al., 1966; Bast and Melampy, 1972); it is barely detectable in the newly formed corpora lutea of pregnancy and pseudopregnancy (Lamprecht et al. 1969; Turolla et al., 1970; Strauss and Stambaugh, 1974). *In partial fulfilment of the requirements Weizmann Institute of Science.
for Ph. D. degree of the Graduate
School of the
214
S. A. Lamprecht et al.
The mechanisms
governing
modulation
of 20a-OH-SDH
activity
are not
fully understood, but there is strong evidence that prolactin suppresses the synthesis of the enzyme in the corpus luteum: pharmacological blockade of pituitary prolactin secretion results in induction of 20a-OH-SDH activity in corpora lutea of pregnant or pseudopregnant rats and the induction is prevented by administration of prolactin (Lamprecht et al., 1969; Hashimoto and Wiest, 1969b). Hashimoto and Wiest (1969a) have followed the appearance of the activity of 20a-OH-SDH in the first generation corpora lutea formed in prepubertal rat ovaries after synchronized ovulation triggered by exogenous pregnant mare’s serum gonadotrophin (PMSG). This technique in inducing formation of corpora lutea has the advantage that growth and development of the newly formed corpora lutea can be studied without the co-existence of corpora lutea in various stages of regression, as found in the ovary of adult cycling rats. Hashimoto and Wiest (1969a) injected 4 I.U. PMSG to Holzmann rats on day 26, leading to ovulation and corpus luteum formation early on day 29. The 20a-OH-SDH activity started to increase in these corpora lutea 4-5 days after the induced ovulation. In vivo administration of prostaglandin F,, (PGF*,) to pregnant and pseudopregnant rats results in premature termination of luteal function (Duncan and Pharriss, 1970; Pharriss et al., 1972) and it has recently been reported that PGF 2a can induce 20a-OH-SDH activity in corpora lutea of pregnant rats (Fuchs and Mok, 1974; Strauss and Stambaugh, 1974). There is evidence that this action of PGF,, is dependent on the degree of maturity of the corpus luteum (Gutknecht et al., 1969; Fuchs and Mok, 1974; Strauss and Stambaugh, 1974). The present study had the following .objectives: (i) to determine, by direct biochemical analysis of 20a-OH-SDH activity, the onset of spontaneous luteolysis in the first generation corpora lutea from PMSG-treated prepubertal rats, (ii) to examine the corpus luteum response to in vivo administration of PGF,, and (iii) to investigate whether prolactin suppresses a PGF,,-induced rise in 20a-OH-SDH activity in this type of corpora lutea.
MATERIALS
AND
METHODS
Animals and treatments Female Sprague-Dawley rats from Anticimex Ltd., Stockholm, Sweden were used. The animals were kept on a regular day and night schedule, and fed a standard diet and water ad libitum.
PGFza and 20a-OH-SDH
275
In order to obtain first generation corpora lutea, 10 I.U. PMSG (Gestyl, N.V. Organon, Oss, The Netherlands) were given subcutaneously to 30-day-old rats between 8 and 10 a.m. Ovulation occurred early in the morning of day 33 with formation of lo-20 corpora lutea per rat. Corpora lutea were excised with microscalpels with the aid of a dissecting microscope and kept in ice-cold 0.15 M NaCl before incubation; those isolated from 33-day-old rats, 6-10 h after ovulation, are designated as one-day-old corpora lutea with consecutive numbering up to 13-day-old corpora lutea derived from 45-day-old rats (fig. 1). Whole ovaries were trimmed of adherent fat and kept in cold saline before assay of 20a-OH-SDH. Three different sets of experiments were performed. In the first set, following administration of the ovulatory dose of PMSG, the rats did not receive any further treatment. Whole ovaries or newly formed corpora lutea were collected at different time intervals and assayed for 20a-OH-SDH activity. subcutaneously In the second set of experiments, PGF 2a was administered (250 ,ug, twice daily at 5 p.m. and 11 p.m.) to rats at various time intervals following the synchronized ovulation. PGFZa, supplied by the Upjohn Company of Canada, Don Mills, Ontario and ON0 Pharmaceutical Co., Ltd., Osaka, Japan, was dissolved in 9.5% ethanol to which was added a Na,CO,-water mixture (0.2 mg/ml) to achieve a final prostaglandin concentration of 0.5 mg/ml. Control rats received injections of 0.5 ml of vehicle. The rats were sacrificed 9 h after the last injection and the corpora lutea examined for 20a-OH-SDH. In a number of rats, PGFza (250 ug) was administered subcutaneously 6 days after the induced ovulation and the animals were divided into three groups: group I received PGFza 6 and 3 h prior to sacrifice, group II 12 and 6 h and group III 18 and 9 h before collection of corpora lutea. In the third set of experiments, 200 ug of ovine prolactin (National Institutes of Health, P-S-3 in 0.9 % saline) was injected concurrently with PGF,, (250 pg) to 39-day-old rats, six days after ovulation. Control rats received PGF,, only. The injections were given 18 and 9 h before the assay of luteal 20a-OH-SDH. The stated potency of the prolactin preparation was 15 I.U./mg; it was free of detectable luteinizing hormone (less than 0.0004 NIH-LH-SI units/mg) and follicle-stimulating hormone (less than 0.009 NIH-FSH-SI units/mg) activity. Reagents and substrates All chemicals and solvents were of analytical grade. 20a-Hydroxypregn4-en-3-one (Ikapharm, Ramat Gan, Israel) was examined by paper and gasliquid chromatography and found to be >98 % pure. NADP+ (Sigma Grade, Sigma Chemical Co., St. Louis, USA) was dissolved in the incubation medium immediately before use.
276
S. A. Lantprech~ et al.
The assay procedure followed closely that of Lamprecht et al. (1969). Tris buffer, 0.1 M (pH 8.0) containing 1 mm01 EDTA, 1 mmol cysteine and 10 mmoles nicotinamide per 1 was used as the medium for homogenization and incubation. The ovaries were removed, trimmed of adherent fat, and the corpora lutea excised. Each sample contained 10-20 corpora lutea derived from a single rat. The pooled corpora lutea were homogenized for 2 min when immersed in crushed ice and the homogenates were spun in a Sorvall refrigerated centrifuge at 15,000 g for 20 min. The supernatant was taken for protein determination according to Lowry et al. (1951). 20a-OH-SDH activity was assayed in 1 cm light path quartz cuvettes. Each cuvette was filled with 2.7 ml of the buffered medium, 1 pmole of NADP dissolved in 0.1 ml of the same medium, and 0.316 nmole of 20uhydroxypregn4-en-3-one dissolved in 0.1 ml ethanol. The cells were then preincubated at 37 “C for 10 min and the reaction was initiated by adding 0.1-0.3 ml of the tissue extract supernatant. NADPH generation was measured by continuously recording the absorbance at 340 nm. Activity of 20cc-OH-SDH was expressed in terms of milliunits (mU) of enzyme activity per mg 15,000 g supernatant fraction protein (mU/mg protein), where one unit of activity was defined as the formation of 1 pmole of NADPH per min at 37 “C under conditions of the assay,
RESULTS Administration of 10 I.U. of PMSG to 30-day-old prepubertal rats initiates the series of events shown in fig. 1. In another series of experiments (Hillensjb et al., 1974) it was shown that the PMSG-injected rats had an endogenous
corpus luteum -b
PM%
.
IOIU
I
I
I
I
I,
I
CL-12
CL-9
CL-6
I
,
I
n
I
I
I
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Rat age (days) Fig. 1. Schematic illustration of the PMSG model. CL-3, CL-6, CL-9 and CL-12 represent 3, 6, 9 and 12-day-old corpora lutea (CL), respectively.
277
PGFza and 20a-OH-SDH
Table 1. 20a-OH-SDH activity in whole ovaries and isolated corpora lutea of different ages from prepubertal rats injected with 10 I.U. PMSG on day 30. 20a-OH-SDH was measured in mU/mg protein, where mU = milliunits of enzyme activity, one unit being equal to the formation of 1 umole NADPH/min at 37 “C. Values are given as means & S.E.M. with the numbers of observations in parenthesis. N.D. = not detectable. Compartment analyzed
Rat age (days)
whole whole corpus corpus corpus corpus corpus
32 33 34-41 42 43 44 45
ovary ovary luteum luteum luteum luteum luteum
Corpus luteum age (days) before ovulation 1 (6-8 h) 2-9 10 11 12 13
20a-OH-SDH (mU/mg protein) N.D. N.D. N.D. 6 and 52 76f15 96 f 32 139 k 29
(2) (5) (4) (5)
release of LH in the afternoon of the 32nd day. The rats presented 12-1.5 ova in the tubal ampulla in the morning of day 33 corresponding to the normal number of ova found in the cycling rat. Whole ovaries collected 48 h after the administration of PMSG and corpora lutea dissected 6-9 h after ovulation were devoid of 20a-OH-SDH activity: l-9day-old corpora lutea also showed no measurable activity (table 1). A spontaneous rise in 20a-OH-SDH activity was observed in lo-day-old corpora lutea and enzyme activity increased progressively in older corpora lutea.
Table 2 Effect of PGFza on 20a-OH-SDH activity in the first generation corpora lutea of different ages from prepubertal rats injected with 10 I.U. PMSG on day 30. Abbreviations are as in table 1. PGFza (250 pg) was injected 18 and 9 h prior to isolation of corpora lutea. 20a-OH-SDH
(mU/mg protein)
Corpus luteum age (days) Control 2
3 4 6 7
N.D. N.D. N.D. N.D. N.D.
PGFz,-treated N.D. N.D. 6 i 0.9 17 + 2 34 i 5
(5) (5) (5)
278
S. A. Lampvecht
0
%a
et al.
plj5. Prolactin
Fig. 2. PGF,, (250 pg) and prolactin (200 pg) were injected concurrently S.C. to 39-day-old PMSG-treated rats, i.e. 6 days after ovulation. Two injections were given, 18 and 9 h prior to isolation of corpora lutea and determination of 20a-OH-SDH activity. There were 9 observations in one group (PGFza alone) and 8 in the other.
In vivo administration of PGF*, (250 pg/rat b.i.d.) resulted in the induction of 20a-OH-SDH activity in 4-day-old corpora lutea. Younger corpora lutea, l-3 days of age, were refractory to this action of PGFza (table 2). The effect of PGF,, was more pronounced in older corpora lutea: in 7-day-old corpora lutea the activity of 20a-OH-SDH was approximately 5-fold higher than in corpora lutea 3 days younger (table 2). The stimulation of 20a-OH-SDH in the corpora lutea was shown to be a rapid process. When PGF,, (250 pg) was given 6 and 3 h, 12 and 6 h, and 18 and 9 h, respectively, prior to isolation of 6-day-old corpora lutea, marked 20a-OH-SDH activity was observed as early as 6 h after the PGF,, injection. This stimulation did not become more pronounced after 12 and 18 h. In vivo administration of prolactin (KC. 200 pg/rat b.i.d.) partially prevented the PGF,,-induced rise of 20a-OH-SDH in the 7-day-old corpora lutea (fig. 2); enzyme activity was reduced by 50%, a difference which is statistically significant (P~0.05).
279
DISCUSSION In the present experiments, the first generation corpora futea had a life-span of lo-13 days, similar to that observed in the corpora lutea of the pseudopregnant rat. 20a-OH-SDH activity appeared spontaneously on day 10, and the enzyme activity increased during the following days. Hashimoto and Wiest (1969a) obtained a single generation of functional corpora lutea after administration of a lower.dose of PMSG (4 I.U.) to prepubertal rats at the age of 26 days. A spontaneous rise of ZOa-OH-SDH activity was found in corpora lutea 4-5 days ofd, at a time when no enzyme activity was found in corpora lutea of our PMSG model. The dose of PMSG used in relation to the normal puberty of the strain may be an important factor in causing this discrepancy. The luteolytic process in our experiments was triggered in 4-9-day-old corpora lutea by administration of PGF,,: activity of 20a-OH-SDH was observed as early as 6 h after administration of the drug. An effect of PGF2, in inducing luteal 20a-OH-SDH has also been observed in pregnant rats, both by determining the progestin pattern in the ovary (Behrmann et al., 1971) and by determination of 20a-OH-SDH activity in isolated corpora lutea and ovaries (Strauss and Stambaugh, 1974; Fuchs and Mok, 1974). The relatively rapid action of PGF,, in stimulating 20a-OH-SDH activity is in marked contrast to the 24-48 h Iatency of the effect of ergocornine, an alkaloid known to induce ZOa-OH-SDH activity in the corpora lutea of the pregnant and pseudopregnant rat (Lamprecht et al., 1969). The hormonal mechanisms involved in triggering the physiological luteolysis in the ovary of the rat are unclear. There is considerable evidence that the non-pregnant uterine endometrium produces a luteolysin in many mammals including the rat (e.g. Pharriss et al., 1972) and there is evidence in favour of prostaglandins as endogenous uterine luteolytic factors in some species (Blatchley and Donovan, 1969; McCracken, 1971). The results of the present study together with earlier investigations (e.g. Pharriss and Wyngarden, 1969; Behrman et al., 1971) show that exogenous PGFza mimics spontaneous luteolysis in the rat. However, factors other than PGFza also influence the onset of luteolysis. It has been suggested that prolactin and LH, the main components of the luteotrophic complex in the rat, may exert Iuteolytic actions, depending on the age and the endocrine status of the corpus luteum and the phase of the reproductive cycle (Rothchild, 1965; Malven, 1969). In our series of experiments, responsiveness to exogenous PGF,, in the first generation corpora lutea was clearly correlated with the age of the corpus luteua. Corpora lutea younger than 3 days did not respond to the action of the PGF2a, and the effect of PGFza in stimulating 20a-OH-SDH activity became morz pronounced
280
with increasing
S. A. Lamprecht
age. The magnitude
of the effect of PGF,,
has also been shown to depend on the age of corpora (Gutknecht et al., 1969; Fuchs and Mok, 1974).
in inducing
et al.
abortion
lutea in the pregnant
rat
An intriguing question is which mechanisms cause acquisition of responsiveness to exogenous PGF,, during a time-span of 24 h (between the 3rd and 4th day). It is possible that the endocrine environment to which the corpus luteum is exposed may play a crucial role in the timing of the acquisition of responsiveness to prostaglandins. It is therefore of interest to note that l-dayold corpora lutea already have receptor sites for LH and PGE, since they can respond to stimulation by either of these substances with an increase in CAMP (Herlitz et al., 1974). The view that prolactin maintains luteal function in the rat has been based, in the past, exclusively on results of biological assays, such as the support of pregnancy (Cutuly, 1941) and deciduomata formation (Evans et al., 1941) or the persistance of vaginal mucification in estrogen-treated, hypophysectomized rats (Astwood, 1941). It has been assumed that these effects reflect a stimulation of luteal progesterone synthesis. There is, however, increasing evidence that prolactin might owe its luteotrophic action mainly to an inhibitory effect on the synthesis of 20cr-OH-SDH and additional ovarian reductases (Lamprecht et al., 1969; Hashimoto and Wiest, 1969b; Zmigrod et al., 1972). The finding of the present study that concurrent administration of prolactin and PGFza resulted in an inhibitory influence on the induction of 20cr-OH-SDH in 7-day-old corpora lutea favours this hypothesis. A similar suppressive effect of prolactin on PGF,.-induced 20a-OH-SDH activity has recently been seen in corpora lutea of pregnancy (Strauss and Stambaugh, 1974). Establishment of pseudopregnancy, formation of deciduomata and direct determination of post-ovulatory secretion of progesterone have provided evidence for the functional capacity of the first set of corpora lutea in the study of Hashimoto and Wiest (1969a). We have not yet explored these parameters of luteal function in our experimental model. Herlitz et al. (1974) have, however, studied the in vitro effect of LH on the CAMP content of the same types of corpora lutea dissected l-7 days after ovulation in PMSG-treated rats. The young corpora lutea showed an extensive response while LLY produced only a minimal increase in CAMP in the old corpora lutea. The reason why aged corpora lutea responded less efficiently to stimulation by LH in vitro is unclear. An attractive hypothesis, although totally speculative, is that this behaviour reflects the initiation of the regression of corpus luteum. Some of the possibilities considered above concerning the development of are equally relevant to the explanation of responsiveness to exogenous PGF,, the dramatic quantitative change in response to LH as a function of corpus
PGF,,
luteum
281
and 20a-OH-SDH
age. These considerations
emphasize
the fact that the corpus luteum
in
its ephemeral existence is an ever-changing biochemical and structural entity, and that it is not meaningful to discuss how the corpus luteum behaves without considering its past history and present environment.
ACKNOWLEDGMENTS We want to thank Professor Hans Lindner for valuable suggestions. We are also indebted to the Endocrinology Study Section of NIH and N.V. Organon for the generous supply of hormones and Ono Pharmaceutical and Upjohn Companies for the gift of PGF,,. Skilled technical assistance was performed by Mrs Anita Sjbgren. The present investigation was supported by grants from the Swedish Medical Research Council (B74-03X-27), U.S. Public Health (2 ROl HD 0279.5), Ford Foundation, Population Council and from the Medical Faculty, University of Goteborg.
REFERENCES Astwood, E. B. (1941) Endocrinology 28, 309. Bast, J. D. and Melampy, R. M. (1972) Endocrinology 91, 1499. Behrman, H. R., Yoshinaga, K., Wyman, H. and Greep, R. 0. (1971) Am. J. Physiol. 221,189. Blatchley, F. R. and Donovan, B. T. (1969) Nature 221, 1065. Cutuly, E. (1941) Proc. Sot. Exptl. Biol. Med. 48, 315. Duncan, G. W. and Pharriss, B. B. (1970) Fed. Proc. 29, 1232. Evans, H. M., Simpson, M. E., Lyon, W. R. and Turpeinen, K. (1941) Endocrinology 28,933. Fuchs, A.-R. and Mok, E. (1974) Biol. Reprod. 10, 24. Gutknecht, G. D., Cornette, J. C. and Pharriss, B. B. (1969) Biol. Reprod. 1, 367. Hashimoto, I. and Wiest, W. G. (1969a) Endocrinology 84, 873. Hashimoto, I. and Wiest, W. G. (1969b) Endocrinology 84, 886. Herlitz, H., Hamberger, L., Rosberg, S. and Ah&n, K. (1974) Acta Endocrinol. 77, 737. Hillensjo, T., Barnea, A., Nilsson, L., Herlitz, H. and Ah&n, K. (1974) Endocrinology 95, 1762. Lamprecht, S. A., Lindner, H. R. and Strauss III, J. F. (1969) Biochim. Biophys. Acta 187,133. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193,265. Malven, P. V. (1969) In: The Gonads, Ed.: K. W. McKerns (Appleton-Century-Crofts, New YorIc) p. 367. McCracken, J. A. (1971) Ann. N.Y. Acad. Sci. 180, 465. Pharriss, B. B. and Wyngarden, L. J. (1969) Proc. Sot. Exptl. Biol. Med. 130, 92. Pharriss, B. B., Tillson, S. A. and Erickson, R. R. (1972) Rec. Prog. Horm. Res. 28, 51. Pupkin, M., Bratt, H., Weisz, J., Lloyd, C. W. and Balogh Jr, K. (1966) Endocrinology 79, 316. Rothchild, I. (1965) Acta Endocrinol. 49, 107. Strauss III, J. F. and Stambaugh, R. L. (1974) Prostaglandins 5, 73.
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Talwalker, P. K., Krahenbuhl, C. and Desaulles, P. A. (1966) Nature 209, 86. Turolla, E., Baldratti, G. and Scrascia, E. (1970) Experientia 26, 418. Wiest, W. G. (1959) J. Biol. Chem. 234, 3115. Wiest, W. G., Kidwell, W. R. and Balogh Jr, K. (1968) Endocrinology 82, 844. Wiest, W. G. and Kidwell, W. R. (1969) In: The Gonads, Ed.: K. W. McKerns (AppletonCentury-Crofts, New York) p. 295. Wilcox, R. B. and Wiest, W. G. (1960) Endocrinology 67, 281. Zmigrod, A., Lindner, H. R. and Lamprecht, S. A. (1972) Acta Endocrinol. 69, 141.