Prolactin binding sites in the male rat liver following castration.

Specific binding sites for prolactin (PRL) have been detected in membrane preparations from the liver of the male rat following castration. The magnitude of the increased binding following castration varied with the age of the animals and with the time after castration. The effect of castration did not appear to be PRL mediated, since increases or decreases of serum PRL levels after pharmacological agents had no effect on PRL binding. The pituitary, however, seems to have a critical role in mediating the increase in PRL binding. Adrenalectomy did not influence the extent of binding of PRL after castration. Testosterone administration, however, completely prevented the increased PRL binding which followed castration. These studies suggest that testosterone has a modulating effect on hepatic PRL binding sites. The maintenance of such binding activity requires not only PRL, but also a functioning pituitary.

A human B-lymphoblastoid cell line produces prolactin.

A variety of cell lines were examined by Northern blot hybridization for the expression of PRL or PRL-related mRNAs. We found that a human B-lymphoblast cell line transcribed a mRNA which hybridized to human PRL cDNA under high stringency conditions. The human lymphoblast cell line of interest is a variant subline of the IM-9 line that we have designated IM-9-P. The lymphoblast-derived PRL mRNA is approximately 150 bases longer than that produced by the human pituitary as determined by Northern blot analysis. IM-9-P PRL was immunoaffinity purified from conditioned medium and found to be identical in mol wt by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to human pituitary PRL. Moreover, IM-9-P PRL is biologically active in the rat Nb2 lymphoma mitogenic assay. Ribonuclease-H digestion of mRNA poly(A) tracts indicated that the size difference between pituitary and IM-9-P PRL transcripts was not due to an elongated poly(A) tail on the lymphoid PRL mRNA. Genomic Southern blot analysis showed no major rearrangements of the PRL gene in IM-9-P cells compared to the parent IM-9 line and human placenta DNA. Thus, it is highly likely that an elongation of the 5' and/or 3' untranslated regions of IM-9-P PRL mRNA account for the size difference with pituitary PRL mRNA. The PRL-producing IM-9-P line was cloned by limiting dilution, and a high PRL-producing clone IM-9-P3 and a non-PRL producer IM-9-P6 were isolated for further analysis. IM-9-P3 cells were found to secrete 40-50 ng PRL/10(6) cells.24 h regardless of cell density. The level of PRL mRNA also remained constant during exponential growth of IM-9-P3 cells. The existence of the PRL-producing IM-9-P3 clone and the IM-9-P6 clone which does not produce PRL as well as the IM-9 progenitor line provides a unique system with which to analyze the molecular mechanism of ectopic human PRL expression.

Decidual-type prolactin expression by the human myometrium.

The human myometrium, in addition to the decidualized endometrium of the late luteal phase and of pregnancy, has been proposed as a second source of uterine PRL, since immunoreactive PRL was found in supernatants from myometrial explant cultures. We demonstrate here that: 1) the human (h) PRL gene is expressed in the myometrium in vivo; 2) myometrial PRL is identical to pituitary hPRL; 3) the encoding transcript differs from pituitary hPRL messenger (m) RNA but is homologous to decidual and IM-9-P3 lymphoid hPRL mRNA; and 4) the expression of myometrial hPRL mRNA is inhibited by progestin. hPRL mRNA was detected in freshly isolated myometrium by Northern blot hybridization and was larger than the pituitary message. Sequence and primer extension analyses revealed that the transcript is identical to pituitary hPRL mRNA downstream of the pituitary cap site and carries an extension of the 5'-untranslated region homologous to that of decidual/IM-9-P3 lymphoid hPRL mRNA. This mRNA species results from alternative transcription initiation and comprises exon 1a of the hPRL gene, which is not transcribed in the pituitary. hPRL mRNA steady state levels and hPRL secretion increased dramatically when myometrial explants were maintained in long term culture. In addition to the 23,000 mol wt form, myometrial explants synthesized a glycosylated hPRL variant (G-hPRL) which was approximately 500 Daltons larger than pituitary G-hPRL but of similar size as lymphoid G-hPRL (26,500). Lactogenic activity of myometrial conditioned medium paralleled that of pituitary hPRL in the Nb2 lymphoma bioassay and was neutralized by the addition of monoclonal antibody to hPRL. hPRL secretion and hPRL mRNA abundance were not affected by estrogen but were markedly reduced by medroxy-progesterone acetate, which was maximally effective at a dose as low as 10(-10) M.

Regulation of prolactin secretion in the human B-lymphoblastoid cell line IM-9-P3 by dexamethasone but not other regulators of pituitary prolactin secretion.

We examined whether the ectopic production of human(h) PRL by the human B-lymphoblastoid cell line IM-9-P3 is under hormonal control. We demonstrate that PRL secretion in this cell line is regulated by dexamethasone but not by other hormones known to modulate PRL secretion in the pituitary or decidua. Dexamethasone caused a reduction of secretion rates to 30% of control values after 3 day paralleled by a decrease in hPRL mRNA levels, without affecting cell viability. Half-life determinations of hPRL mRNA in control and dexamethasone-treated cells revealed a reduction of t1/2 from 16 to 4 h. PRL secreted by IM-9-P3 cells did not control its own secretion in an autocrine loop, nor did it serve as an autocrine growth factor.

In vivo effects of antisera to prolactin receptors in female rats.

Antisera generated in guinea pigs against partially purified PRL receptors derived from rabbit mammary glands were tested for their effect on PRL binding and the biological effects of PRL in vitro and in vivo. Several dilutions of either normal guinea pig serum or anti-PRL receptor serum were incubated in vitro with homogenates of rat ovaries. Although specific binding of PRL was inhibited as much as 90% by the anti-serum, normal guinea pig serum had little effect. There was no inhibition of binding of LH and FSH to ovaries by these antisera. When these antisera were administered to normal cycling rats, the most pronounced effect was an increase in the number of corpora lutea, presumably due to the prevention of the luteolytic effect of PRL. When one of these antisera was given to adult rats immediately after parturition, weight gain of their pups decreased significantly, possibly reflecting a decrease of milk yield. Thus, passive immunization of animals with anti-PRL receptor sera modifies the actions of PRL on some of its target tissues.

Phorbol ester stimulates progesterone production by isolated bovine luteal cells.

The tumor promoter, phorbol 12-myristate-13-acetate (PMA), is known to modulate the response of several steroidogenic tissues presumably by activating a Ca++- and phospholipid-dependent protein kinase (protein kinase C). The presence of this kinase has been demonstrated in bovine corpus luteum, although its role in steroidogenesis by these cells is unknown. We report here the effects of PMA on progesterone production by the enzymically dispersed bovine luteal cells in vitro. PMA (1-50 nM) produced a dose- and time-related increase in progesterone production by the luteal cells. The maximum stimulation was achieved with 10 nM PMA. Higher concentrations of PMA led to a decline of steroidogenesis close to the basal level. A nonpromoting derivative, 4 alpha-phorbol 12,13-didecanoate had no effect. The PMA-induced stimulation of progesterone production was not associated with a change in the cAMP level. PMA added together with suboptimal doses of human CG, 8Br-cAMP, cholera toxin, or forskolin significantly increased the amount of progesterone produced. PMA as well as human CG-induced steroidogenesis was sensitive to cycloheximide inhibition. The conversion of exogenous pregnenolone or 25-hydroxycholesterol to progesterone was not altered by PMA. We conclude that PMA at nanomolar concentrations is able to stimulate progesterone production by bovine luteal cells and that the site of action of PMA is distal to the formation of cAMP but before the formation of pregnenolone. The observed effects of PMA in luteal cells are probably linked to its ability to activate protein kinase C, since a diacylglycerol could mimic the steroidogenic action of PMA.

Hyperprolactinemic anovulatory syndrome.

The functional status of the hypothalamo-pituitary-gonadal axis was investigated in 127 women with anovulatory disease. Radioimmunoassayable circulating LH, FSH, and prolactin concentrations were measured. An attempt was made to localize the functional lesion by utilizing the following criteria: 1. Hypothalamic function: a) clomiphene test based upon hormonal parameters; b) recording of the pulsatile LH fluctuation (spiking) and of basal FSH. 2. Pituitary function: determination of the gonadotropin reserve by means of a standardized LRH test. 3. Ovarian function: a) measurement of plasma E2 and progesterone levels by RIA; b) gestagen bleeding test. All patients had amenorrhea of up to 14 years duration. A total of 17 hyperprolactinemic patients (13.4%) was found. Eight of these patients never experienced galatorrhea, in 7 only transient galactorrhea was reported, and in 2 cases galactorrhea persisted. All hyperprolactinemic patients were found to be clomiphene non-responders as well as nonspikers. The pituitary LH reserve varied from practically none to normal. Baseline LH was low whereas that of FSH was normal. In accordance with this observation E2 levels, with two exceptions, were found to be in the lower range of normal female concentrations. Thus, all but two patients exhibited gestagen withdrawal bleeding. In conclusion, the hyperprolactinemic anvoluatory syndrome is not necessarily associated with galactorrhea. In all cases of amenorrhea syndromes with or without galactorrhea, hyperprolactinemia should be excluded as it is very often associated with anovulation. The hyperprolactinemic anovulatory syndrome includes the following features: 1. gestagen withdrawal bleeding. 2. subnormal to normal E2 levels. 3. clomiphene nonresponsiveness. 4. LH-hypogonadotropism. 5. lack of LH secretory episodes. 6. FSH-normogonadotropism.

Human pituitary gonadotropin index. I. Standardized LRH test criteria for evaluation of functional amenorrhea.

LRH test were carried out by giving 339 amenorrheic women and 74 normally menstruating volunteers an intravenous injection of 25 mug LRH (Hoe 471). Plasma LH and FSH were measured by RIA in two laboratories (Tuebingen and Ulm) using two standard reference preparations: LER 907 and 2nd IRP-HMG. The average conversion factors between the two standard preparations were calculated at 5.0 for LH and 25.0 for FSH. Furthermore, the estradiol-17beta levels were measured in 139 out of the 339 patients immediately before and 60 minutes after LRH injection. Taking the episodic and cyclic plasma gonadotropin fluctuations into consideration a shorthand system classifying the gonadotropin baseline (BI-BIV) and LH responses to 25 mug LRH (R0-R2) has been established and is referred to as Human Pituitary Gonadotropin Index (HPGI). It is possible to achieve reproducible gonadotropin results in two different laboratories using two different standard reference preparations. Two separate, randomly selected groups of amenorrheic women were found to have the same percent distribution of the HPGI. A correlation coefficient of r equal 0.67 between basal and LRH stimulated plasma LH levels does not sufficiently characterize the individual LH response behavior. A significant increase of plasma LH and FSH within the test period (60') reveals that the iv administration of 25 mug LRH represents an adequate dose for the LRH test in women. The HPGI which characterizes the functional state of gonadostat, may become a useful diagnostic index for evaluating women with anovulatory disease before, during, and after therapy.

Testosterone production by mouse Leydig cells is stimulated in vitro by atrial natriuretic factor.

The synthetic atrial peptides, rat atrial natriuretic peptide, atriopeptin I and atriopeptin II, stimulated testosterone production by mouse Leydig cells in a time- and concentration-dependent manner. The maximum stimulation of the steroidogenesis in response to the peptides was 6-10-fold over the basal level, as compared with 20-24-fold stimulation obtained with saturating concentrations of hCG. The stimulation of steroidogenesis by the most potent peptide, atriopeptin II, was markedly enhanced in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, suggesting an involvement of cyclic nucleotides. However, neither basal nor hCG-stimulated levels of cAMP were altered by the peptide, though testosterone production in response to submaximal concentrations of hCG was increased in the presence of atriopeptin II. The nature of the second messenger involved and the mechanism of action of the atrial peptides may be elucidated by further research in progress.

Prolactin (PRL) mRNA from human decidua differs from pituitary PRL mRNA but resembles the IM-9-P3 lymphoblast PRL transcript.

The expression of the human prolactin (hPRL) gene is normally restricted to the anterior pituitary and the decidualized endometrium of the uterus. The human B-lymphoblastoid cell line IM-9-P3 ectopically expresses a PRL mRNA which is about 150 nucleotides larger than its pituitary counterpart even though the mature protein products appear identical. In the present study we show that human decidual and IM-9-P3 PRL mRNAs are similar in size, both being elongated relative to the pituitary transcript. The size difference persisted after removal of the 3' end poly(A) tract, whereas the PRL precursors synthesized in a cell-free translation system using mRNAs from the three tissues showed identical apparent molecular weights upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The elongation could therefore not be attributed to differences in the protein coding region or in the degree of polyadenylation and is suggested to reside in the 5' untranslated region of the molecule.

Phorbol ester stimulates prolactin release but reduces prolactin mRNA in the human B-lymphoblastoid cell line IM-9-P3.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated prolactin (PRL) release from the PRL producing human B-lymphoblastoid cell line IM-9-P3 within 30 min with an EC50 of 5 x 10(-9) M. Increased release was entirely attributable to a loss from intracellular PRL pools. No change in hPRL mRNA was observed during 8 h of exposure to TPA. Prolonged exposure of the cells to 2 x 10(-7) M TPA, however, led to a maximal reduction of hPRL mRNA levels after 24 h and a subsequent recovery by 72 h. Secretory rates followed a corresponding kinetic. The relative abundance of c-myc mRNA was not affected, although a persistent inhibition of cellular proliferation occurred upon chronic exposure to TPA. The addition of dibutyryl cAMP caused a minor transient increase in hPRL secretion by 35% after 1 h.

Prolactin secretion during pregnancy and puerperium: response to metoclopramide and interactions with placental hormones.

Prolactin (PRL) response to an intravenous administration of metoclopramide (10 mg) was examined during normal pregnancy and puerperium. Basal PRL and the metoclopramide-induced increase of PRL increased gradually during pregnancy. This was paralleled by serum estradiol, estriol, and progesterone levels. A positive correlation between serum progesterone and metoclopramide-induced PRL concentrations was found at week 36 of pregnancy. In lactating women, metoclopramide always induced higher increases of PRL than did suckling stimulation on the seventh day postpartum. The PRL responses to suckling and metoclopramide were significantly correlated with each other, but no correlation was found between placental steroid levels throughout pregnancy, PRL levels after parturition, and total milk production during seven days postpartum.

Prolactin stimulation tests: different response patterns after bromocriptine, lisuride, and metergoline treatment of puerperal women.

To elucidate different mechanisms by which bromocriptine, lisuride, and metergoline may inhibit prolactin (PRL) secretion and lactation in puerperal women, the PRL secretion patterns were examined by means of a stimulation test using an intravenous bolus of metoclopramide. After seven and 14 days of treatment, no significant difference in basal serum PRL levels was observed. However, women subjected to metergoline treatment had significantly higher responses of PRL to metoclopramide as compared with those treated with either bromocriptine or lisuride. Thus, the PRL-lowering mechanism of metergoline appears to be different from those of bromocriptine and lisuride.

Treatment of hyperprolactinemic amenorrhea with Metergoline.

The ergoline derivative, Metergoline, in a dosage of 4 to 24 mg/day, was administered for one to eight months to 42 patients with hyperprolactinemic amenorrhea. Mean serum prolactin (PRL) concentrations before treatment were 91.2 ng/mL in the patients with functional hyperprolactinemia (N = 29) and 256.9 ng/mL in the patients with pituitary tumor (N = 13). Within four weeks, Metergoline treatment reduced these PRL concentrations to 39.5 ng/mL and 82.9 ng/mL, respectively. In this study Metergoline treatment resulted in restoration of menstruation in a total of 37 patients; 28 patients ovulated, and eight became pregnant. It is considerably more effective in functional hyperprolactinemia than in hyperprolactinemia caused by adenoma.

Continuous, combined hormone replacement: randomized comparison of transdermal and oral preparations.

OBJECTIVE: To compare two new transdermal, continuous, combined formulations and an oral regimen of hormone replacement therapy (HRT) with respect to endometrial hyperplasia, bleeding patterns, and climacteric symptoms in postmenopausal women. METHODS: This was a randomized, open, parallel-group trial during 1 year in 441 postmenopausal women who received either a 10-cm2 patch of 0.025 mg estradiol (E2) and 0.125 mg norethisterone acetate, a 20-cm2 patch of 0.05 mg E2 and 0.25 mg norethisterone acetate twice weekly, or tablets of 2 mg E2 and 1 mg norethisterone acetate once daily. The efficacy variables were frequency of endometrial hyperplasia after 1 year of treatment, number of bleeding and spotting days from the fourth to sixth treatment months, relief of climacteric symptoms, and tolerability. RESULTS: The frequency of endometrial hyperplasia was no more than 2% after 1 year of treatment in all groups. One case of simple hyperplasia was detected among the women treated with 10-cm2 patches and two among those treated with oral HRT. From the fourth to sixth treatment months, amenorrhea occurred in 73%, 47%, and 66% of the women in the 10-cm2, 20-cm2, and oral HRT groups, respectively. The 10-cm2 patches and oral treatment were associated with fewer bleeding days than were the 20-cm2 patches (P<.001). During the last 3 months of the treatment year, amenorrhea was found in 100 subjects (86%) for 10-cm2 patches, 61 (65%) for 20-cm2 patches, and in 85 (79%) for oral HRT. All treatments alleviated the climacteric symptoms to a comparable extent. CONCLUSION: In postmenopausal women, 10-cm2 patches relieved climacteric symptoms and prevented endometrial hyperplasia at least as effectively as oral HRT. Amenorrhea was induced early in a high percentage of women using 10-cm2 patches and oral HRT, and these therapies seemed to be convenient, effective, and safe for estrogen deficiency symptoms in postmenopausal women.

Morphometric analysis of the endometrium of infertile patients in relation to peripheral hormone levels.

In an attempt to identify factors that may be responsible for reproductive failure, we compared endometrial biopsies taken from infertile patients during the luteal phase of spontaneous cycles (n = 18) with those taken after ovarian stimulation (n = 18). Morphometric analyses were performed and compared with peripheral estradiol (E2) and progesterone levels at the time of supposed implantation. In stimulated cycles the number of glands per square millimeter was positively correlated to the E2 level. The morphometric data point to a measurable difference between the study groups, indicating an insufficient secretory transformation of the tissue in infertile women when compared with a group of fertile women. The observations suggest that hormonal stimulation occasionally results in impaired development of the endometrial glands.

Patterns of serum-luteinizing hormone surges in stimulated cycles in relation to injections of human chorionic gonadotropin.

Endogenous-luteinizing hormone (LH) surges may complicate the management of in vitro fertilization cycles. To investigate the effects of LH surges after hormonal stimulation 53 IVF cycles were analyzed by assessing LH levels three times daily until egg collection. In 43% the LH rise started before the planned exogenous trigger for ovulation was given, in 11% the rise occurred simultaneously with and in 45% after the injection of human chorionic gonadotropin. Three main patterns of serum LH surges were identified: (A) low-LH tonus with straight increase to maximum; (B) low tonus with elevation before straight increase; (C) high tonus with large variations but no prominant peak. These patterns were not related to the follicular estradiol increase, luteal steroid concentrations or resulting pregnancy rates.

Preliminary report about a modified gonadotropin (human menopausal gonadotropin/human chorionic gonadotropin) treatment in infertile patients with premature luteinization.

Premature luteinization is a frequent phenomenon observed in infertile women undergoing human menopausal gonadotropin/human chorionic gonadotropin (hMG/hCG) therapy for corpus luteum insufficiency or anovulatory cycles. By inducing hyperprolactinemia in these women with sulpiride, we intended to create a dysfunctional state in these women, which supposedly induces a better reaction to hMG/hCG therapy. The rationale behind this combined treatment was the observation that hyperprolactinemic amenorrheic patients have a much higher pregnancy rate under hMG/hCG treatment than the above group. Three cases are reported in detail, illustrating the altered ovarian response to combined sulpiride-hMG/hCG treatment. Of 60 infertile women with repeated premature luteinization, 12 conceived after sulpiride-hMG/hCG therapy. Their expected pregnancy rate would have been very low (5% or less) if conventional hMG/hCG treatment had been continued.

Morphometric characteristics of endometrial biopsies after different types of ovarian stimulation for infertility treatment.

OBJECTIVE: To investigate whether various types of ovarian stimulation induce differences in endometrial development at the midluteal phase in infertile women. DESIGN: Assessment of stromal and glandular compartments in endometrial biopsies using morphometric criteria. SETTING: Institute for Hormone and Fertility Research, Hamburg, Germany. PATIENTS: The study included 18 women after treatment with human menopausal gonadotropin (hMG)/human chorionic gonadotropin (hCG) (group I), 23 women after clomiphene citrate (CC)/hMG/hCG treatment (group II), and 12 women after CC stimulation (group III). INTERVENTIONS: Endometrial biopsies and blood samples were taken simultaneously in the early to midluteal phase. To assess the time of ovulation, hormone analysis and regular checks by ultrasonography were performed. MAIN OUTCOME MEASURES: Morphometric evaluation of glandular and stromal structures revealed an impaired endometrial development after various treatment protocols. CONCLUSION: Ovarian stimulation in infertile women results in most cases in an elevation of steroid levels; however, the occurrence of an inadequate endometrial development might have an unfavorable influence on the outcome of implantation. Therefore, these findings may be of importance to the choice of treatment for infertility.

Epimestrol in treatment of inadequate luteal progesterone secretion.

Seventeen infertile normoprolactinemic women with luteal phase defects were treated with epimestrol. In ten patients, normalization of the impaired luteal phase was achieved. Under epimestrol treatment, periovulatory estradiol concentrations (1077.3 +/- 121.5 pmoles/liter versus 612.7 +/- 64.2 pmoles/liter; mean +/- SEM; P < 0.01) and cervical scores (10.8 +/- 0.3 versus 7.9 +/- 0.38; mean +/- SEM; P < 0.01) were improved, and luteal progesterone (60.0 +/- 9.1 nmoles/liter versus 19.98 +/- 3.14 nmoles/liter; mean +/- SEM; P < 0.001) and estradiol secretioon (813.1 +/- 101.1 pmoles/liter versus 581.9 +/- 73.7 pmoles/liter; mean +/- SEM; P < 0.05) were significantly increased in those women with normalization of the luteal phase as compared with the seven patients in whom epimesterol was without effect. Prolactin levels were elevated in all patients after epimestrol therapy (P < 0.05). Basal LH levels and LH-RH stimulated levels improved following administration of epimestrol (P < 0.01) in the 10 women with normal luteal phases. FSH levels were not significantly affected. Two patients became pregnant. No side effects were noted.