J. Spona

OVULATION INHIBITION WITH DIFFERENT DOSES OF LEVONORGESTREL* AND OTHER PROGESTOGENS: CLINICAL AND EXPERIMENTAL INVESTIGATIONS

Abstract. Model systems were evaluated, which can be used to determine biological activities of progestogens at various cell levels of the anterior pituitary gland. in addition, various progestogens at different dose levels were tested in females to evaluate the ovulation inhibition dose. the association of progestogens with the cytosol receptor of the adenohypophysis of the rat was studied in vitro. Sucrose gradient centrifugation analysis revealed material sedimenting in the 4‐5 S and 6‐7 S region, resp., and both binding proteins were found to specifically bind progestogens. a small displacement by cortisol was observed in the 4‐5 S area. Relative affinity constants were estimated for various progestogens by incubation of cytosol samples with radioactive labeled progestins, and a close correlation between affinity and biological activity was recorded. Adult female rats seven days after castration were used to study the influence of various progestogens on LH‐RH stimulated gonadotropin release. Levonorgestrel was found to augment LH‐RH provoked LH release in vivo, whereas both basal as well as LH‐RH stimulated FSH serum levels were noted to be impaired at higher doses of the steroid. Similarly, δ15‐levonorgestrel was observed to increase the response of LH to LH‐RH. on the other hand, δ15‐levonorgestrel greatly augmented basal as well as LH‐RH induced FSH serum levels up to a dose of 10 μg. At 100 μg of δ15‐levonorgestrel FSH serum levels were suppressed again. But, only 1/3 of the levonorgestrel dose was necessary to produce these effects, i. e. δ15‐levonorgestrel was found ca. 3‐times more potent than the native progestogen. Experiments performed in a cell culture system of anterior pituitary cells revealed an impairment of LH‐RH stimulated gonadotropin release. LH and FSH release was suppressed to 40 and 50%, resp., at ED50 of LH‐RH in the presence of 3×10−8M levonorgestrel. LH and FSH release was reduced to 50 and 70%, resp., when anterior pituitary cells were incubated with LH‐RH in the presence of 3×10−8M δ15‐levonorgestrel. Thus, cell culture experiments also suggested that the potency of the δ15‐derivative of levonorgestrel was ca. 3‐times more potent. Analysis of patterns of cervical scores and karyopyknotic index as well as of serum levels of 17β‐estradiol, progesterone, LH and FSH revealed a borderline ovulation inhibition dose of levonorgestrel and δ15‐levonorgestrel at 50 and 30 μg, respectively. Cyproterone acetate, which is used in an oral contraceptive to treat acne and hirsutism was found to inhibit ovulation at a borderline dose of 1 mg. the present data combine to suggest that all model system are efficient to evaluate new progestogens, and that δ15‐levonorgestrel exerts a some 3‐times greater biological activity than levonorgestrel itself. Thus, a dose reduction of the progestogen component in oral contraceptives seems to be possible.

LH-RH interaction with the pituitary plasma membrane is affected by sex steroids

Variation of pituitary responsiveness to synthetic LH-RH was observed during different phases of the menstrual cycle [l-3] . Recently changes of in vitro response to LH-RH were reported for pituitaries of male and female rats, respectively, at different ages [4,5] . Furthermore, LH-RH stimulated release of gonadotropins could be provoked by estrogen pretreatment in females suffering from amenorrhoea who did not respond to LH-RH prior to the steroid application [6] . Additionally, LH-RH stimulated-LH release was strikingly higher in normal pubertal children than in children of prepubertal ages [7] . These fmdings suggested that changes in hypophyseal responsiveness to LH-RH were affected by estradiol and/or progesterone. Additional evidence for this suggestion was obtained by recent in vitro experiments, which showed that 17 Sestradiol and progesterone could modulate responses of adenohypophyses to LH-RH [8] . Very recently LH-RH was shown to be bound to isolated plasma membranes of rat anterior pituitaries [9]. LH-RH-receptor interaction with the anterior pituitary plasma membrane was characterized and found to be a temperature and pH dependent process [lo] . Data currently available strongly suggest that two distinct LH-RH binding sites are present in the pituitary receptor system of intact rats. But, one binding site of the receptor system could no longer be detected if binding experiments were performed with plasma membranes obtained from adenohypophyses of chronically ovariectomized rats [ 1 l] . This observation and data on in vitro stimulation of gonadotropin release by

Ontogeny of 17β-estradiol-binding protein in the female rat hypothalamus and anterior pituitary

A line of evidence suggests that estrogen exerts a feedback on the hypothalamus-pituitary-axis. Experiments in the human indicate that two sets of estrogen sensitive neurones exist in the hypothalamus [ 11, of which one set is stimulated by estrogen to secrete LH-RH, whereas the other set of neurones secreting LH-RH is postulated to be sensitive to estrogen de& ciency. Similar mechanisms have been postulated for the rat [2-41. Previous observations [5,6] led to suggest that estrogen was an important factor involved in the maturation process of the brain-pituitary unit and in the regulation of the onset of puberty. Binding of 17fl-estradiol to the pituitary and brain was studied by autoradiographic techniques [7] and a specific, limited-capacity 17/I-estradiol-binding protein of the cytoplasm of the rat hypothalamus was described recently [8-131. Maximal responses to LH-RH were reported during pre-pubertal ages in rats [ 14,151 and changes were found in the in vitro responsiveness to LH-RH of pituitaries of rats at different ages [ 16,171. Furthermore, it was shown that 17/3-estradiol and other steroid hormones modulated LH-RH stimulated gonadotropin release [18,19]. The interaction of 17/3-estradiol with intracellular receptor proteins is an acknowledged primary event, which presumably mediates physiological activities in estrogen-responsive tissues. This process has been exten-

LH-RH—Receptor interaction is inhibited by Des-His—2-Des-Gly—10-LH-RH—ethylamide

Luteinizing hormone releasing hormone (LH-RH) is a hypothalamic hormone which stimulates the secretion of pituitary hormone(s) which regulate ovulation [I] . The elucidation of the amino acid sequence (pGlu-His-Trp-Ser-Tyr-Gly-Leu-ArgPro-Gly-NHz) of LH-RH [l] has enabled a considerable number of related peptides to be synthesized and examined for biological activities. Much data has accumulated on the structure-activity relationship of this hormonal peptide. The results of these studies suggested that the active site of LH-RI-l was located in the N-terminal region of the molecule, that is, the pGlu and the His residues played important roles in the hormonal activity [2-S]. Furthermore, studies on analogs of LH-RH lacking the N-terminal pGlu ring structure suggested that the CO-NHCHCOgroup was the minimum necessary part of the pGlu residue to exhibit biological activity. Recently several analogs of LH-RH with enhanced LH and FSH releasing activity were synthesized [6-lo] . Interest in derivatives of LH-RH has arisen, primarily, because of the possibility of finding analogs of LH-RH which would inhibit the release of gonadotropins instead of stimulating them. This may form the basis of a new contraceptive method. Part of the mechanism of action of LH-RH involves the interaction of the hormone with pituitary cell receptors [ 111, and the binding of LH-RH to the adenohypophysis could be located at the level of the plasma membrane [12-131 An important criterion for an inhibitor of LH-RH actions would be that it should effectively compete with the hormone for pituitary receptor sites. We were therefore prompted to report on data concern-

PROGESTERONE RECEPTOR IN THE RAT ANTERIOR PITUITARY Transformation and nuclear translocation

Cytoplasmic estrogen [ 1-3], glucocorticoid [3,4] and androgen receptors [3,5,6] have been detected in the rat pituitary. But, evidence for specific progesterone receptor has been conflicting [3,7]. Recently, progestin binding sites were detected in the rat anterior pituitary [8,9]. Previously, it was shown that progestins modulate according to their biological potency LH-RHstimulated gonadotropin release in vivo [ 10]. In addition, progesterone was observed to influence LH-RHstimulated LH and FSH release in vitro [11,12] and suppression of gonadotropin release is a well-acknowledged mechanism of action of hormonal contraceptives. These observations combine to suggest that progesterone acts at least partially at the pituitary level in regulating gonadotropin release. Whether or not specific progesterone receptors are present in the hypophysis is essential for the understanding of the mechanism of action of progesterone on the brain. Since estrogen was reported to induce cytoplasmic progesterone receptors in the animal uterus [ 13,14] it is likely that a progesterone receptor is detectable in neural tissues containing estrogen receptors. Ontogeny of the rat anterior pituitary estrogen-binding protein was described [15] and nuclear translocation of the estrogen receptor was reported [16]. The aim of the present investigation was to study the association of a progestin with the cytosol receptor in the rat anterior hypophysis and the transformation of the receptor complex in vitro. In addition, experiments were designed to investigate the nuclear translocation of the cytosol receptor complex in vitro. We were prompted to report these data since they add further evidence that receptor translocation is a primary step in the molecular events of progesterone action.

Nuclear translocation of the rat pituitary cytosol 17β-estradiol receptor

The association of 17fl-estradiol with an 8 S binding protein in the cytoplasm is a generally acknowledged primary step in the cellular action of the steroid hormone. The complex crosses the nuclear membrane either before or after conformational change of the protein to a 5 S species [ I] . The presence of an estrogen receptor in the pituitary is well documented [2-51 and the ontogeny of the pituitary cytosol 17P-estradiol receptor was described recently [6]. In addition, the actions of estrogen on the hypothalamus-pituitary axis have been studied extensively. Little is known, however, about the molecular mechanisms of LH-RH stimulated gonadotropin release modulated by 17flestradiol at the pituitary level. The‘present experiments were designed to study the translocation of the pituitary estrogen-receptor complex into the nucleus in rats under in vivo and in vitro conditions. Furthermore, the effect of castration on the translocation process in the hypothalamus and pituitary of female rats was investigated. We were prompted to report these data since they add further evidence that estrogen stimulated translocation of the pituitary 17flestradiol receptor from the cytosol to the nucleus is a primary event in the mechanism of action of this steroid. 2. Materials and methods

A monospecific antibody population in cross-reacting anti-human placental lactogen serum

Several years ago a close structural similarity between human growth hormone (HGH) and human platen tal lactogen (HPL), also known as human chorionic somatomammotropin (HCS), was proposed on the basis of physiological and immunological data [ l-31 , and could be contirrned by several studies on the amino acid sequences of the two hormones [4, 51 Recently we could show that despite this high degree of structural similarity a cross-reacting anti-human growth,hormone serum may be resolved into a monospecific antibody population [6] . A very recent publication [ 71 indicates that the regions of homology seem to include more than 90% of all sequence positions. This finding prompts us to report our experiments on the isolation of a monospecific antibody population from a cross-reacting anti-human placental lactogen serum.

Resolution of a cross-reacting anti-human growth hormone serum into a monospecific antibody population.

Antisera raised in animals against human growth hormone (HGH) have previously been shown to crossreact with human placental lactogen (HPL) [l] . Recently, a large homology in amino-acid sequence between HGH and HPL was described [2,3]. However, differences in the magnitude of the biological activity of the two hormones were observed [4]. The striking similarities between a pituitary and placental protein is intriguing and lead us to investigate whether in this model system for cross-reacting antisera monospecific antibody populations directed against HGH only can be isolated. We wish to report a resolution of antibodies in an anti-HGH serum into cross-reacting and monospecific populations by affinity chromatography using a specific HPL-immunoadsorbent.

Norethisterone does not inhibit rat testis Δ5- 3β-hydroxysteroid dehydrogenase

A key step in the biosynthesis of progesterone and of androgens is the production of A4-3-ketosteroids from A5-3/I-hydroxysteroids [ 11. This enzymatic conversion is a two step process in which dehydrogenation is followed by isomerization 121. The first step is rate limiting, and the enzyme which mediates the dehydrogenation is A5-3&hydroxysteroid dehydrogenase (EC 1.1.1.145); it can be found in extracts of Pseudomonas testosteroni [3] and is localized in the microsomes and mitochondria of all steroid synthesizing organs such as adrenal, ovary, placenta and testis [4]. and stored at 20” C. The cyanoketone used was 2~ cyano4,4,17cu~trimethylandrost-5-en-17fi-ol-3-one. /3-Nicotinamide adenine dinucleotide was purchased from Sigma Chem. Co. (St Louis MO). All other chemicals were of analytical grade and were obtained from Sigma, Boehringer or Merck AG (Darmstadt).

Steroidhormon-Rezeptoranalyse an malignen Melanomen

Der potentielle Einflus von Steroidhormonen auf das biologische Verhalten des malignen Melanoms wurde auf Grund klinischer Beobachtungen bereits seit langem diskutiert. So war bekannt, das vor der Pubertat die Inzidenz des Tumors uberaus selten ist. Bei Frauen tritt er hingegen am haufigsten zu Beginn der Menopause auf. Eine rasche Disseminierung des Melanoms wahrend der Schwangerschaft und spontane Regression nach der Entbindung sind beschrieben; andererseits haben Frauen eine bessere Krankheitsprognose als Manner.