SERUM FOLLICLE-STIMULATING HORMONE, LUTEINIZING HORMONE AND PROGESTERONE CONCENTRATIONS IN PSEUDOPREGNANT RATS TREATED WITH MEDROXYPROGESTERONE ACETATE
GREEF, J. DULLAART AND G. H. ZEILMAKER Department of Endocrinology, Growth and Reproduction, and* Department of Anatomy, Erasmus University, P.O. Box 1738, Rotterdam, The Netherlands
W. J.
DE
(Received 19
December
1975)
SUMMARY
Pseudopregnant rats were treated early in pseudopregnancy with 1 or 10 mg medroxyproacetate (MPA). Serum FSH, LH and progesterone concentrations were deteron mined days 2\p=n-\20of pseudopregnancy in treated and control rats. The mean duration of pseudopregnancy was 13\m=.\5days in the control animals, but when animals were treated with 1 mg MPA a dioestrous period of 21\m=.\4days was observed. A period with leucocytic vaginal smears of at least 2 months was observed after treatment with gesterone
10 mg MPA. Injection with MPA on day 3 of pseudopregnancy did not affect the serum FSH concentrations during the subsequent days. The progesterone pattern was alike in the three groups of animals, i.e. the duration of the activity of the corpora lutea was similar in all groups. However, 10 mg MPA slightly lowered progesterone concentrations on days 4\p=n-\8of pseudopregnancy. In the saline-treated rats, LH concentrations decreased from days 2\p=n-\5,and remained low until they increased after day 11 of pseudopregnancy. This increase was delayed until day 20 in the animals treated with 1 mg MPA, and was not observed in the animals treated with 10 mg MPA. It is argued that the increase of LH concentration at the end of pseudopregnancy is not instrumental in the decrease of peripheral progesterone concentration but rather that the decrease in the progesterone concentration leads to the increase in the LH concentration.
INTRODUCTION
An inverse relationship between the concentrations of progesterone and luteinizing hormone (LH) is observed in the rat during pseudopregnancy and pregnancy : low LH concentrations are found when high progesterone concentrations are present (Morishige, Pepe & Roth¬ child, 1973; Welschen, Osman, Dullaart, de Greef, Uilenbroek & de Jong, 1975). At the end of pseudopregnancy a decrease in progesterone concentration is observed (Bartosik &
Szarowski, 1973; de Greef & Zeilmaker, 1974; Pepe & Rothchild, 1974), together with an increase in LH concentration (Bast & Melampy, 1972; Welschen et al. 1975). The question
arises whether the increase of LH concentration is instrumental in the induction of the irre¬ versible decrease in luteal progesterone secretion, which is associated with an increase in luteal 20a-hydroxyprogesterone secretion (functional luteolysis; see Malven, 1969; Bartosik & Szarowski, 1973). In an attempt to answer this question, the effect of administration of a
long-acting synthetic progestagen [6a-methyl-17-acetoxypregn-4-ene-3,20-dione, medroxyprogesterone acetate; a potent anti-ovulation compound assumed to suppress pituitary gon¬ adotrophin secretion (Labhsetwar, 1966; Rifkind, Kulin, Cargille, Rayford & Ross, 1969; Dickmann, 1973)] on progesterone and LH levels during pseudopregnancy was measured. MATERIALS AND METHODS
(RxU) Fx hybrid rats, weighing 200-230 g, were used in these studies. The animals were exposed to a schedule of 14 h light and 10 h darkness, constant room temperature (22-24 °C), and standard laboratory diet and tap water ad libitum. Vaginal smears were taken 6 days per week between 09.00 and 10.00 h. Animals were used only after three con¬ secutive 5-day cycles had been recorded. Pseudopregnancy was induced by sterile mating and the day on which the copulation plug was observed was designated as day 0 of pseudo¬ pregnancy. The duration of pseudopregnancy was determined by counting the number of days from metoestrus until the first metoestrous day of the ensuing cycle. Medroxyprogesterone acetate (MPA, Depo-Provera, Upjohn) was diluted with 0-9 % NaCl solution to make suspensions containing 1 or 10 mg MPA/0-5 ml. In the first experi¬ ment, 1 or 10 mg MPA were injected subcutaneously between 17.00 and 18.00 h on day 3 of pseudopregnancy, and in the second experiment 10 mg MPA were given on day 1 of pseudo¬ pregnancy between 16.00 and 18.00 h. In both experiments the control rats were injected subcutaneously with 0-5 ml saline. Blood was collected within 1-2 min after removal of the animal from its cage between 09.00 and 11.00 h by puncturing the orbital plexus under light ether anaesthesia. From each animal 2-3 blood samples of about 1 ml were taken with at least 2 days between successive bleedings. After collection the blood samples were allowed to clot in a refrigerator before centrifugation. Serum samples were stored at —18 °C until assayed. Serum LH and follicle-stimulating hormone (FSH) concentrations were estimated by radioimmunoassay as described previously (Welschen et al. 1975). NIAMDD-rat-LH RP-1 and NIAMDD-rat-FSH RP-1 were used as standards. Serum progesterone concen¬ trations were determined with a radioimmunoassay (de Jong, Baird & van der Molen, 1974) as described previously (de Greef, Dullaart, & Zeilmaker, 1975; Meijs-Roelofs, de Greef & Uilenbroek, 1975). Medroxyprogesterone acetate did not seem to interfere with this assay system: 50pg caused only a 10 % displacement of radioactive progesterone from the anti¬ body. Furthermore, when 10 mg MPA were given to ovariectomized rats no increase in progesterone concentration was found. The results were analysed by a two-factor analysis of variance for the overall analysis, by Student's t test for the independent samples, and by Wilcoxon matched-pairs signed-ranks test for the related samples. A difference was considered to be significant if the double-tail probability was < 0-05. All results are expressed as means ± s.e.m., unless otherwise is indicated. Adult
RESULTS
Serum FSH, LH and progesterone concentrations during pseudopregnancy after administration of medroxyprogesterone acetate on day 3 ofpseudopregnancy Rats were treated on day 3 of pseudopregnancy with saline, 1 or 10 mg MPA. In rats treated with saline the duration of pseudopregnancy was 13-5+1-2 days (mean±S.D.; 35). Treatment with 1 mg MPA prolonged the period of vaginal dioestrus to 21-4+1-8 days 38). After a dose of 10 mg MPA, a much longer period with leucocytic (mean ± s.d. ; smears was observed: in all animals leucocytes were observed in the vaginal smears for a period of 2 months (n 37). The FSH concentrations measured are shown in Fig. 1. During days 4-10 the mean serum =
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Day of pseudopregnancy Fig. 1. Serum FSH concentrations (means ± s.e.m.) in pseudopregnant rats after treatment on day 3 of pseudopregnancy with saline (white bars, 6), 1 mg medroxyprogesterone acetate (hatched 6), or 10 mg medroxyprogesterone acetate (black bars, « 5). bars, =
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Fig. 2. Serum LH concentrations (means ± s.e.m.) in pseudopregnant rats after treatment on day 3 of pseudopregnancy with saline (white bars), 1 mg medroxyprogesterone acetate (hatched bars), or 10 mg medroxyprogesterone acetate (black bars). The number of animals is given on top of the bars. For comparison, non-injected values are shown for days 2 and 3.
FSH concentrations were between 90 and 110 ng FSH RP-l/ml, and no differences were found between the three groups of rats. Concentrations of FSH in saline-treated rats were significantly lower at day 12 of pseudopregnancy than at days 8 and 10 (P < 0-05). In the rats tteated with 1 mg MPA, lower mean FSH concentrations seemed to be present at day 20 than at days 16 or 18; however, these differences were not significant (P > 0-05). In saline-treated rats serum LH concentrations decreased significantly from days 2-5 (P < 0-025) and remained low until day 11 ; thereafter, LH concentrations increased again (Fig. 2). Medroxyprogesterone acetate suppressed serum LH concentrations in the pseudo¬ pregnant animals, the higher dose being more effective (overall analysis for days 4-10: < 0-005, when compared with control rats). An 1 mg MPA, < 0-05 and 10 mg MPA, in increase LH concentrations was observed from days 18 to 20 in rats treated with 1 mg MPA (P < 0-025), but in rats treated with 10 mg MPA no increase was observed. In control rats serum progesterone concentrations increased significantly from 23 ng/ml on day 2 to 85 ng/ml on day 5 (P < 0-005), remained relatively constant until day 8, and decreased thereafter to a concentration of 15 ng/ml on day 13 (Fig. 3). The same profile was 90
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Day of pseudopregnancy 3. Serum concentrations progesterone Fig. (means ± s.e.m.) in pseudopregnant rats after treatment on day 3 with saline (white bars), 1 mg medroxyprogesterone acetate (hatched bars), or 10 mg medroxyprogesterone acetate (black bars). The number of animals is indicated on top of the bars. For comparison, non-injected values are shown for days 2 and 3. found in animals treated with MPA (Fig. 3), and no significant difference was found bet¬ ween control rats and animals injected with 1 mg MPA. The mean progesterone concentra¬ tions on days 4-8, however, were slightly, but significantly, lower in animals treated with 10 mg MPA than in the saline-treated animals (overall analysis: < 0-025). Serum progesterone concentrations during pseudopregnancy after administration of medroxy¬ progesterone acetate on day 1 ofpseudopregnancy The possibility exists that MPA has a direct effect on the activity of the corpora lutea, in¬ ducing a decrease in progesterone secretion only after a certain period. Therefore, MPA was
given on day 1 of pseudopregnancy. Animals treated with 10 mg MPA on day 1 of pseudopregnancy, however, had a progesterone profile identical to that seen in animals injected with 10 mg MPA on day 3 of pseudopregnancy. Therefore, the decrease in proges¬ terone concentration after day 8 of pseudopregnancy in the animals treated with MPA is not also
related to the time of MPA administration. Furthermore, after treatment with 10 mg MPA on day 1 a slight suppression of the progesterone concentration was observed on days 4-8 of pseudopregnancy (overall analysis: < 0-01, when compared with animals given saline on day 1 of pseudopregnancy). discussion
The present study confirms the observation of Dickmann (1973) that a prolonged period of vaginal dioestrus is found in rats treated with MPA. It is likely that ovulations are absent during this period of vaginal leucocytosis (Labhsetwar, 1966). As judged from the vaginal smear data, the duration of the luteal phase seemed to be prolonged. Our observations indi¬ cated, however, that the prolonged period of vaginal dioestrus induced by MPA was not associated with a persistent progesterone secretion. Therefore, the evidence of vaginal smears alone is not sufficient to diagnose the existence of a luteal phase (Zeilmaker, 1964). Treatment with MPA prevented the increase in LH concentrations normally seen on days 11 and 12 of pseudopregnancy. When 1 mg MPA was given, the increase in LH concentra¬ tion was noted on day 20, which was 1-3 days before an oestrous smear was observed in these animals. After administration of 10 mg MPA, LH concentrations remained low during the period of observation, and no sign of return to cyclicity was observed. In the present study no differences in progesterone concentrations were observed when MPA was given on days 1 or 3 of pseudopregnancy. Apparently, a fixed relationship between the time of MPA administration and the decrease of progesterone concentrations around day 9 of the luteal phase does not exist. This finding makes it unlikely that MPA has a direct effect on the corpora lutea. In spite of low LH values in the animals treated with MPA, functional luteolysis, as indi¬ cated by peripheral progesterone concentrations, occurred at the same time in all groups. Therefore, it is reasonable to suggest that functional luteolysis at the end of pseudopregnancy is not dependent on an increase in LH concentration. It is more likely that the increase in LH concentration at the end of pseudopregnancy is caused by the decrease of progesterone in the circulation, as was suggested previously by Welschen et al. (1975). Whether the ab¬ sence of an increase in LH concentration delays the occurrence of structural luteolysis re¬ mains to be established. It was demonstrated that a dose of 10 mg MPA slightly suppressed serum progesterone concentrations in the pseudopregnant rat. A depression of progesterone concentration during the luteal phase was also observed in women treated with MPA after ovulation (Johansson, 1971). A similar finding was made when three other progestagens (norethiste¬ rone, norgestrel or chlormadinone acetate) were given to women after ovulation (Johansson, 1971). From that study it can be concluded that the action of these progestagens is either directly on the ovary or via the hypothalamus-hypophysis system influencing gonadotro¬ phin secretion. The finding that human chorionic gonadotrophin (HCG) increased plasma progesterone concentrations in women treated with norgestrel or norethisterone (Johansson, 1971) suggests that the depression of progesterone concentrations following treatment with these progestagens is brought about by a decrease of gonadotrophin secretion. Support for this concept is derived from the studies in which it was shown that MPA suppresses urinary excretion of LH in man (Kupperman & Epstein, 1962; Laron, Rumney, Rat & Naji, 1963; Rifkind et al. 1969). This decrease of LH concentrations could lead to a decrease of proges¬ terone synthesis in the corpus luteum. The effects of MPA on progesterone concentration
noticed in the present study may also be attributed to the decrease of LH concentration since LH has, under certain conditions, a stimulatory effect on progesterone synthesis in the rat (Armstrong, O'Brien & Greep, 1964; Armstrong, Miller & Knudsen, 1969). However, the possibility that MPA suppresses progesterone secretion by direct action on the ovary cannot be excluded entirely. In-vitro studies indicate that a compound related to MPA (chlormadinone acetate) inhibits the A5-3/?-hydroxysteroid dehydrogenase activity in porcine ovarian tissue (Aakvaag, 1970). Serum FSH concentrations were unaltered after MPA treatment. In rats treated with saline, a decrease of FSH concentration was observed on day 12 of pseudopregnancy, and in the animals treated with 1 mg MPA a small decrease in FSH concentrations at the end of the dioestrous phase was also found. This decline of FSH concentration towards the end of pseudopregnancy is probably caused by the increase in oestradiol-17/5 concentration resulting from the increase in LH concentration (Welschen et al. 1975).
The authors wish to thank Professor H. J. van der Molen for the gift of the antiserum used in the progesterone assay, and the NIAMDD for the materials used in the LH and FSH assays. The cooperation of Miss M. M. Duin, Mrs A. A. de la Fosse-Kuijpers and Miss H. P. Boesberg during this study is greatly appreciated. The authors are indebted to Pro¬ fessor J. Moll for his valuable criticism during the preparation of the manuscript. We thank Dr P. E. Schenck for helpful suggestions during the statistical analysis. This work was supported in part by the Organization of Medical Research in the Netherlands (FUNGO). references
Aakvaag, A. (1970). Steroid formation in porcine ovarian tissue in vitro. Acta Endocrinologica 65, 261-272. Armstrong, D. T., Miller, L. S. & Knudsen, K. A. (1969). Regulation of lipid metabolism and progesterone production in rat corpora lutea and ovarian interstitial elements by prolactin and luteinizing hormone. Endocrinology 85, 393-401. Armstrong, D. T., O'Brien, J. & Greep, R. O. (1964). Effects of luteinizing hormone on progestin biosynthe¬ sis in the luteinized rat ovary. Endocrinology 75, 488-500. Bartosik, D. & Szarowski, D. H. (1973). Progravid phase of the rat reproductive cycle: day-to-day changes in peripheral plasma progestin concentrations. Endocrinology 92, 949-952. Bast, J. D. & Melampy, R. M. (1972). Luteinizing hormone, prolactin and ovarian 20a-hydroxysteroid dehydrogenase levels during pregnancy and pseudopregnancy in the rat. Endocrinology 91, 1499-1505. Dickmann, . (1973). Short- and long-term effects of a single injection of depo-medroxyprogesterone acetate (Provera) on the vaginal smear, ovulation and mating in the rat. Journal of Reproduction and Fertility 32, 447-451. de Greef, W. J., Dullaart, J. & Zeilmaker, G. H. (1975). Serum luteinizing hormone, follicle-stimulating hormone, prolactin and progesterone concentrations and follicular development in the pseudopregnant rat after unilateral ovariectomy. Journal of Endocrinology 66, 249-256. de Greef, W. J. & Zeilmaker, G. H. (1974). Blood progesterone levels in pseudopregnant rats: effects of partial removal of luteal tissue. Endocrinology 95, 565-571. Johansson, E. D. B. (1971). Depression of the progesterone levels in women treated with synthetic gestagens after ovulation. Acta Endocrinologica 68, 779-792. de Jong, F. H., Baird, D. T. & van der Molen, H. J. (1974). Ovarian secretion rates of oestrcgens, andro¬ gens and progesterone in normal women and in women with persistent ovarian follicles. Acta Endocrinologi¬ ca 77, 575-587. Kupperman, H. S. & Epstein, J. A. (1962). Medroxyprogesterone acetate in the treatment of constitutional sexual precocity. Journal of Clinical Endocrinology and Metabolism 22, 456-458. Labhsetwar, A. P. (1966). Mechanism of action of medroxyprogesterone (17a-acetoxy-6a-methyl proges¬ terone) in the rat. Journal of Reproduction and Fertility 12, 445-451. Laron, Z., Rumney, G., Rat, L. & Naji, N. (1963). Effects of 17a-hydroxy-6a-methylprogesterone acetate (Depo-Provera) on urinary gonadotrophins and oestrogens in man. Acta Endocrinologica 44, 75-80. Malven, P. V. (1969). Hypophysial regulation of luteolysis in the rat. In The gonads, pp. 367-382. Ed K. W. McKerns. New York : Appleton-Century-Crofts. Meijs-Roelofs, . . ., de Greef, W. J. & Uilenbroek, J. Th. J. (1975). Plasma progesterone and its rela¬ tionship to serum gonadotrophins in immature female rats. Journal of Endocrinology, 64, 329-336.
Morishige, W. K., Pepe, G. J. & Rothchild, I. (1973). Serum luteinizing hormone, prolactin and proges¬ terone levels during pseudopregnancy in the rat. Endocrinology 92, 1527-1530. Pepe, G. J. & Rothchild, I. (1974). A comparative study of serum progesterone levels in pregnancy and various types of pseudopregnancy in the rat. Endocrinology 95, 275-279. Rifkind, A. B., Kulin, H. E., Cargille, C. M., Rayford, P. C. & Ross, G. T. (1969). Suppression of urinary excretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) by medroxyprogesterone acetate. Journal of Clinical Endocrinology and Metabolism 29, 506-513. Welschen, R., Osman, P., Dullaart, J., de Greef, W. J., Uilenbroek, J. Th. J. & de Jong, F. H. (1975). Levels of follicle-stimulating hormone, luteinizing hormone, oestradiol-17/? and prcgesterone, and follicular growth in the pseudopregnant rat. Journal of Endocrinology 64, 37-47. Zeilmaker, G. H. (1964). Aspects of the regulation of corpus luteum function in androgen-sterilized female rats. Acta Endocrinologica 46, 571-579.
GREEF, J. DULLAART AND G. H. ZEILMAKER Department of Endocrinology, Growth and Reproduction, and* Department of Anatomy, Erasmus University, P.O. Box 1738, Rotterdam, The Netherlands
W. J.
DE
(Received 19
December
1975)
SUMMARY
Pseudopregnant rats were treated early in pseudopregnancy with 1 or 10 mg medroxyproacetate (MPA). Serum FSH, LH and progesterone concentrations were deteron mined days 2\p=n-\20of pseudopregnancy in treated and control rats. The mean duration of pseudopregnancy was 13\m=.\5days in the control animals, but when animals were treated with 1 mg MPA a dioestrous period of 21\m=.\4days was observed. A period with leucocytic vaginal smears of at least 2 months was observed after treatment with gesterone
10 mg MPA. Injection with MPA on day 3 of pseudopregnancy did not affect the serum FSH concentrations during the subsequent days. The progesterone pattern was alike in the three groups of animals, i.e. the duration of the activity of the corpora lutea was similar in all groups. However, 10 mg MPA slightly lowered progesterone concentrations on days 4\p=n-\8of pseudopregnancy. In the saline-treated rats, LH concentrations decreased from days 2\p=n-\5,and remained low until they increased after day 11 of pseudopregnancy. This increase was delayed until day 20 in the animals treated with 1 mg MPA, and was not observed in the animals treated with 10 mg MPA. It is argued that the increase of LH concentration at the end of pseudopregnancy is not instrumental in the decrease of peripheral progesterone concentration but rather that the decrease in the progesterone concentration leads to the increase in the LH concentration.
INTRODUCTION
An inverse relationship between the concentrations of progesterone and luteinizing hormone (LH) is observed in the rat during pseudopregnancy and pregnancy : low LH concentrations are found when high progesterone concentrations are present (Morishige, Pepe & Roth¬ child, 1973; Welschen, Osman, Dullaart, de Greef, Uilenbroek & de Jong, 1975). At the end of pseudopregnancy a decrease in progesterone concentration is observed (Bartosik &
Szarowski, 1973; de Greef & Zeilmaker, 1974; Pepe & Rothchild, 1974), together with an increase in LH concentration (Bast & Melampy, 1972; Welschen et al. 1975). The question
arises whether the increase of LH concentration is instrumental in the induction of the irre¬ versible decrease in luteal progesterone secretion, which is associated with an increase in luteal 20a-hydroxyprogesterone secretion (functional luteolysis; see Malven, 1969; Bartosik & Szarowski, 1973). In an attempt to answer this question, the effect of administration of a
long-acting synthetic progestagen [6a-methyl-17-acetoxypregn-4-ene-3,20-dione, medroxyprogesterone acetate; a potent anti-ovulation compound assumed to suppress pituitary gon¬ adotrophin secretion (Labhsetwar, 1966; Rifkind, Kulin, Cargille, Rayford & Ross, 1969; Dickmann, 1973)] on progesterone and LH levels during pseudopregnancy was measured. MATERIALS AND METHODS
(RxU) Fx hybrid rats, weighing 200-230 g, were used in these studies. The animals were exposed to a schedule of 14 h light and 10 h darkness, constant room temperature (22-24 °C), and standard laboratory diet and tap water ad libitum. Vaginal smears were taken 6 days per week between 09.00 and 10.00 h. Animals were used only after three con¬ secutive 5-day cycles had been recorded. Pseudopregnancy was induced by sterile mating and the day on which the copulation plug was observed was designated as day 0 of pseudo¬ pregnancy. The duration of pseudopregnancy was determined by counting the number of days from metoestrus until the first metoestrous day of the ensuing cycle. Medroxyprogesterone acetate (MPA, Depo-Provera, Upjohn) was diluted with 0-9 % NaCl solution to make suspensions containing 1 or 10 mg MPA/0-5 ml. In the first experi¬ ment, 1 or 10 mg MPA were injected subcutaneously between 17.00 and 18.00 h on day 3 of pseudopregnancy, and in the second experiment 10 mg MPA were given on day 1 of pseudo¬ pregnancy between 16.00 and 18.00 h. In both experiments the control rats were injected subcutaneously with 0-5 ml saline. Blood was collected within 1-2 min after removal of the animal from its cage between 09.00 and 11.00 h by puncturing the orbital plexus under light ether anaesthesia. From each animal 2-3 blood samples of about 1 ml were taken with at least 2 days between successive bleedings. After collection the blood samples were allowed to clot in a refrigerator before centrifugation. Serum samples were stored at —18 °C until assayed. Serum LH and follicle-stimulating hormone (FSH) concentrations were estimated by radioimmunoassay as described previously (Welschen et al. 1975). NIAMDD-rat-LH RP-1 and NIAMDD-rat-FSH RP-1 were used as standards. Serum progesterone concen¬ trations were determined with a radioimmunoassay (de Jong, Baird & van der Molen, 1974) as described previously (de Greef, Dullaart, & Zeilmaker, 1975; Meijs-Roelofs, de Greef & Uilenbroek, 1975). Medroxyprogesterone acetate did not seem to interfere with this assay system: 50pg caused only a 10 % displacement of radioactive progesterone from the anti¬ body. Furthermore, when 10 mg MPA were given to ovariectomized rats no increase in progesterone concentration was found. The results were analysed by a two-factor analysis of variance for the overall analysis, by Student's t test for the independent samples, and by Wilcoxon matched-pairs signed-ranks test for the related samples. A difference was considered to be significant if the double-tail probability was < 0-05. All results are expressed as means ± s.e.m., unless otherwise is indicated. Adult
RESULTS
Serum FSH, LH and progesterone concentrations during pseudopregnancy after administration of medroxyprogesterone acetate on day 3 ofpseudopregnancy Rats were treated on day 3 of pseudopregnancy with saline, 1 or 10 mg MPA. In rats treated with saline the duration of pseudopregnancy was 13-5+1-2 days (mean±S.D.; 35). Treatment with 1 mg MPA prolonged the period of vaginal dioestrus to 21-4+1-8 days 38). After a dose of 10 mg MPA, a much longer period with leucocytic (mean ± s.d. ; smears was observed: in all animals leucocytes were observed in the vaginal smears for a period of 2 months (n 37). The FSH concentrations measured are shown in Fig. 1. During days 4-10 the mean serum =
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Day of pseudopregnancy Fig. 1. Serum FSH concentrations (means ± s.e.m.) in pseudopregnant rats after treatment on day 3 of pseudopregnancy with saline (white bars, 6), 1 mg medroxyprogesterone acetate (hatched 6), or 10 mg medroxyprogesterone acetate (black bars, « 5). bars, =
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Fig. 2. Serum LH concentrations (means ± s.e.m.) in pseudopregnant rats after treatment on day 3 of pseudopregnancy with saline (white bars), 1 mg medroxyprogesterone acetate (hatched bars), or 10 mg medroxyprogesterone acetate (black bars). The number of animals is given on top of the bars. For comparison, non-injected values are shown for days 2 and 3.
FSH concentrations were between 90 and 110 ng FSH RP-l/ml, and no differences were found between the three groups of rats. Concentrations of FSH in saline-treated rats were significantly lower at day 12 of pseudopregnancy than at days 8 and 10 (P < 0-05). In the rats tteated with 1 mg MPA, lower mean FSH concentrations seemed to be present at day 20 than at days 16 or 18; however, these differences were not significant (P > 0-05). In saline-treated rats serum LH concentrations decreased significantly from days 2-5 (P < 0-025) and remained low until day 11 ; thereafter, LH concentrations increased again (Fig. 2). Medroxyprogesterone acetate suppressed serum LH concentrations in the pseudo¬ pregnant animals, the higher dose being more effective (overall analysis for days 4-10: < 0-005, when compared with control rats). An 1 mg MPA, < 0-05 and 10 mg MPA, in increase LH concentrations was observed from days 18 to 20 in rats treated with 1 mg MPA (P < 0-025), but in rats treated with 10 mg MPA no increase was observed. In control rats serum progesterone concentrations increased significantly from 23 ng/ml on day 2 to 85 ng/ml on day 5 (P < 0-005), remained relatively constant until day 8, and decreased thereafter to a concentration of 15 ng/ml on day 13 (Fig. 3). The same profile was 90
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Day of pseudopregnancy 3. Serum concentrations progesterone Fig. (means ± s.e.m.) in pseudopregnant rats after treatment on day 3 with saline (white bars), 1 mg medroxyprogesterone acetate (hatched bars), or 10 mg medroxyprogesterone acetate (black bars). The number of animals is indicated on top of the bars. For comparison, non-injected values are shown for days 2 and 3. found in animals treated with MPA (Fig. 3), and no significant difference was found bet¬ ween control rats and animals injected with 1 mg MPA. The mean progesterone concentra¬ tions on days 4-8, however, were slightly, but significantly, lower in animals treated with 10 mg MPA than in the saline-treated animals (overall analysis: < 0-025). Serum progesterone concentrations during pseudopregnancy after administration of medroxy¬ progesterone acetate on day 1 ofpseudopregnancy The possibility exists that MPA has a direct effect on the activity of the corpora lutea, in¬ ducing a decrease in progesterone secretion only after a certain period. Therefore, MPA was
given on day 1 of pseudopregnancy. Animals treated with 10 mg MPA on day 1 of pseudopregnancy, however, had a progesterone profile identical to that seen in animals injected with 10 mg MPA on day 3 of pseudopregnancy. Therefore, the decrease in proges¬ terone concentration after day 8 of pseudopregnancy in the animals treated with MPA is not also
related to the time of MPA administration. Furthermore, after treatment with 10 mg MPA on day 1 a slight suppression of the progesterone concentration was observed on days 4-8 of pseudopregnancy (overall analysis: < 0-01, when compared with animals given saline on day 1 of pseudopregnancy). discussion
The present study confirms the observation of Dickmann (1973) that a prolonged period of vaginal dioestrus is found in rats treated with MPA. It is likely that ovulations are absent during this period of vaginal leucocytosis (Labhsetwar, 1966). As judged from the vaginal smear data, the duration of the luteal phase seemed to be prolonged. Our observations indi¬ cated, however, that the prolonged period of vaginal dioestrus induced by MPA was not associated with a persistent progesterone secretion. Therefore, the evidence of vaginal smears alone is not sufficient to diagnose the existence of a luteal phase (Zeilmaker, 1964). Treatment with MPA prevented the increase in LH concentrations normally seen on days 11 and 12 of pseudopregnancy. When 1 mg MPA was given, the increase in LH concentra¬ tion was noted on day 20, which was 1-3 days before an oestrous smear was observed in these animals. After administration of 10 mg MPA, LH concentrations remained low during the period of observation, and no sign of return to cyclicity was observed. In the present study no differences in progesterone concentrations were observed when MPA was given on days 1 or 3 of pseudopregnancy. Apparently, a fixed relationship between the time of MPA administration and the decrease of progesterone concentrations around day 9 of the luteal phase does not exist. This finding makes it unlikely that MPA has a direct effect on the corpora lutea. In spite of low LH values in the animals treated with MPA, functional luteolysis, as indi¬ cated by peripheral progesterone concentrations, occurred at the same time in all groups. Therefore, it is reasonable to suggest that functional luteolysis at the end of pseudopregnancy is not dependent on an increase in LH concentration. It is more likely that the increase in LH concentration at the end of pseudopregnancy is caused by the decrease of progesterone in the circulation, as was suggested previously by Welschen et al. (1975). Whether the ab¬ sence of an increase in LH concentration delays the occurrence of structural luteolysis re¬ mains to be established. It was demonstrated that a dose of 10 mg MPA slightly suppressed serum progesterone concentrations in the pseudopregnant rat. A depression of progesterone concentration during the luteal phase was also observed in women treated with MPA after ovulation (Johansson, 1971). A similar finding was made when three other progestagens (norethiste¬ rone, norgestrel or chlormadinone acetate) were given to women after ovulation (Johansson, 1971). From that study it can be concluded that the action of these progestagens is either directly on the ovary or via the hypothalamus-hypophysis system influencing gonadotro¬ phin secretion. The finding that human chorionic gonadotrophin (HCG) increased plasma progesterone concentrations in women treated with norgestrel or norethisterone (Johansson, 1971) suggests that the depression of progesterone concentrations following treatment with these progestagens is brought about by a decrease of gonadotrophin secretion. Support for this concept is derived from the studies in which it was shown that MPA suppresses urinary excretion of LH in man (Kupperman & Epstein, 1962; Laron, Rumney, Rat & Naji, 1963; Rifkind et al. 1969). This decrease of LH concentrations could lead to a decrease of proges¬ terone synthesis in the corpus luteum. The effects of MPA on progesterone concentration
noticed in the present study may also be attributed to the decrease of LH concentration since LH has, under certain conditions, a stimulatory effect on progesterone synthesis in the rat (Armstrong, O'Brien & Greep, 1964; Armstrong, Miller & Knudsen, 1969). However, the possibility that MPA suppresses progesterone secretion by direct action on the ovary cannot be excluded entirely. In-vitro studies indicate that a compound related to MPA (chlormadinone acetate) inhibits the A5-3/?-hydroxysteroid dehydrogenase activity in porcine ovarian tissue (Aakvaag, 1970). Serum FSH concentrations were unaltered after MPA treatment. In rats treated with saline, a decrease of FSH concentration was observed on day 12 of pseudopregnancy, and in the animals treated with 1 mg MPA a small decrease in FSH concentrations at the end of the dioestrous phase was also found. This decline of FSH concentration towards the end of pseudopregnancy is probably caused by the increase in oestradiol-17/5 concentration resulting from the increase in LH concentration (Welschen et al. 1975).
The authors wish to thank Professor H. J. van der Molen for the gift of the antiserum used in the progesterone assay, and the NIAMDD for the materials used in the LH and FSH assays. The cooperation of Miss M. M. Duin, Mrs A. A. de la Fosse-Kuijpers and Miss H. P. Boesberg during this study is greatly appreciated. The authors are indebted to Pro¬ fessor J. Moll for his valuable criticism during the preparation of the manuscript. We thank Dr P. E. Schenck for helpful suggestions during the statistical analysis. This work was supported in part by the Organization of Medical Research in the Netherlands (FUNGO). references
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