Expression of steroidogenic acute regulatory protein in the human corpus luteum throughout the luteal phase.

The expression of the steroidogenic acute regulatory protein (StAR) in the human corpus luteum (CL) was examined throughout the luteal phase. The primary 1.6-kb StAR transcript was in greater abundance in early (3.1-fold) and mid (2.2-fold) luteal phase CL compared with late luteal phase CL. The larger StAR transcript (4.4 kb) was found in early and midluteal phase CL, but was not detected in late luteal phase specimens. Mature StAR protein (30 kDa) was present in lower amounts within late CL compared with early and midluteal phase CL. The StAR preprotein (37 kDa) was also detected in greater abundance in early and midluteal CL. Immunohistochemistry revealed that StAR staining was most prominent in thecal-lutein cells throughout the luteal phase. The intensity of the signal for StAR exhibited significant changes throughout the luteal phase, being most intense during the midluteal phase and least during the late luteal phase. Plasma progesterone concentrations were highly correlated (r = 0.73 and r = 0.79) with luteal expression of the preprotein and mature StAR isoforms, respectively, throughout the luteal phase. To examine the LH dependency of StAR expression, the GnRH antagonist, Cetrorelix, was administered during the midluteal phase. Cetrorelix caused a decline in serum LH levels within 2 h, which, in turn, caused a pronounced decline in plasma progesterone within 6 h. The StAR 4.4-kb transcript was not detectable, and the 1.6-kb transcript was reduced by approximately 50% within 24 h of Cetrorelix treatment. The mature 30-kDa StAR protein level declined approximately 30% after Cetrorelix treatment. We conclude that 1) StAR mRNA and protein are highly expressed in early and midluteal phase CL; 2) StAR protein is present in both thecal-lutein and granulosa-lutein cells throughout the luteal phase; 3) StAR protein levels in the CL are highly correlated with plasma progesterone levels; 4) declining StAR mRNA and protein levels are characteristic of late luteal phase CL; and 5) suppression of LH levels during the midluteal phase results in a marked decline in plasma progesterone and a diminished abundance of StAR transcripts in the CL without a corresponding significant decline in StAR protein. Collectively, these data are consistent with the idea that StAR gene expression is a key determinant of luteal progesterone during the normal menstrual cycle. However, the pharmacologically induced withdrawal in the midluteal phase of LH support diminishes luteal progesterone output by mechanisms others than reduced StAR protein levels.

Endocrine and paracrine-autocrine regulation of the human corpus luteum during the mid-luteal phase.

Human corpora lutea undergo an extremely rapid period of growth, development and regression during the course of non-fertile cycles. The tissue consists of steroidogenic (parenchymal) and non-steroidogenic (stromal) cells. In women and other primates, steroid hormone production by corpora lutea depends on the presence of pituitary-derived LH. Nevertheless, there is also intra-luteal regulation of steroid synthesis. Steroidogenic luteal cells and non-steroidogenic cells interact via endocrine and paracrine pathways, and by contact-dependent pathways (gap junctions). Thus, hormones and locally produced factors including steroids, growth factors, cytokines, reactive oxygen species and nitric oxide may modulate luteal lifespan. The factors regulating regression and rescue of the corpus luteum are not understood completely. This review describes the expression of two representative intragonadal peptides that may influence luteal regression (interleukin 1 beta) and luteal rescue (steroidogenic acute regulatory protein).

Interleukin-1beta (IL-1beta) is a modulator of human luteal cell steroidogenesis: localization of the IL type I system in the corpus luteum.

The present investigation examined the effect of interleukin-1beta (IL-1beta) on progesterone production by human luteal cells and the expression and localization of the IL-1 system in the human corpus luteum (CL). Luteal cells were isolated from corpora lutea collected throughout the luteal phase. After dispersion, luteal cells were treated with a panel of monoclonal antibodies directed to leukocyte-specific molecules. The leukocytes were isolated with immunomagnetic beads. Leukocyte-free luteal cells exhibited greater steroidogenic responsiveness to hCG toward the end of the luteal phase. The treatment of mixed luteal cells (total luteal cells) with IL-1beta inhibited by 60% hCG-stimulated progesterone production. Interestingly, the treatment of leukocyte-free luteal cells with IL-1beta did not affect progesterone production. In addition, the treatment of mixed luteal cells with monoclonal antibodies against IL-1 receptor type I (IL-1RtI) resulted in a 2.5-fold increase in the hCG-supported progesterone production. IL-1RtI and IL-1 receptor antagonist were localized by immunohistochemistry in both somatic and immune cells of the CL. Flow cytometric analysis indicated that both nonleukocyte luteal cells and leukocyte-luteal cells exhibited IL-1Rt-I positive cells, representing 56% and 31% of the total luteal cells, respectively. However, 13% of nonleukocyte luteal cells did not express IL-1Rt-I. Northern analysis demonstrated the presence of the 5.1-kb IL-1RtI messenger ribonucleic acid transcript in CL of different ages. RT-PCR indicated that both leukocyte-free luteal cells and luteal leukocytes express IL-1RtI messenger ribonucleic acid. We conclude that 1) luteal leukocytes have an inhibitory effect on hCG-stimulated progesterone production; 2) IL-1beta inhibits hCG-stimulated progesterone production only in mixed luteal cell cultures, indicating that leukocytes mediate the effect; 3) the somatic and immune cells of the CL are sites of action and expression of the IL-1 system; and 4) interaction between the steroidogenic and immune cells of the CL suggests a functional intraovarian role for IL-1beta in CL physiology.

Luteal leukocytes are modulators of the steroidogenic process of human mid-luteal cells.

Flow cytometry analysis of luteal cells revealed that an important proportion of these cells are leukocytes. The percentage of leukocytes was higher in the early (42 +/- 4) and late (35 +/- 3) luteal phases than in the mid-luteal (24 +/- 2) phase. However, the proportion of macrophages did not differ between the luteal stages. The flow cytometric properties correlated with cellular size and granularity were not reliable as discriminators of luteal cell subpopulations. Therefore, to assess the contribution of luteal leukocytes, these cells were completely removed from luteal cell suspensions (total cells), by a negative selection procedure (immunomagnetic separation). The functional role of leukocytes in mid-luteal steroidogenesis was assessed, in total as well as leukocyte-depleted cells. Progesterone production was found to have increased 2.2-fold in leukocyte-depleted cell cultures, in comparison with total cells under basal conditions. However, the response to human chorionic gonadotrophin (HCG) was 36% lower under the latter conditions. Oestradiol production was not significantly modified under basal or HCG-treated conditions. In leukocyte-depleted cells, the concentration of interleukin (IL)-1beta decreased 5-fold in comparison with total cell cultures, suggesting that leukocytes are the principal source of IL-1beta. In summary, the results of the present investigation suggest functional interactions between the immune system and steroidogenic cells of the human corpus luteum.

Hormonal profile, endometrial histology and ovarian ultrasound assessment during 1 year of nomegestrol acetate implant (Uniplant).

The present study assesses the endocrinological, endometrial histology and vaginal ultrasound profiles of nomegestrol acetate subdermal implant users at varying times after insertion. Follicle stimulatory hormone, luteinizing hormone, oestradiol, progesterone, vaginal ultrasound assessment of the ovaries and the histological dating of the endometrium were serially assessed for a period of 50 days immediately after the insertion, and after at 6 months and 12 months of use. The endocrinological results of this prospective observational clinical trial indicated that 75% of the cycles across the study period in Uniplant users were anovulatory, 63% showing development of a persistent non-luteinized follicle. Anovulatory cycles devoid of follicular development were seen primarily in the first months after Uniplant insertion. Ovulatory cycles represented 25% of the Uniplant cycles. Inadequate luteal phase or disregulation of follicular growth was a common feature of ovulatory cycles. In conclusion, these findings suggest that the contraceptive mechanisms of a single nomegestrol acetate subdermal implant involve prevention of follicular growth, development of a persistent non-luteinized follicle, inadequate luteal phase and disruption of the endometrial architecture.

Localization of insulin-like growth factor (IGF-I) and IGF-I receptor expression in human corpora lutea: role on estradiol secretion.

OBJECTIVE: To determine whether insulin-like growth factor (IGF-I) and its receptors are expressed by human corpus luteum (CL) and to establish the effect of IGF-I on E2 biosynthesis in human luteal cell cultures. DESIGN: Middle corpora lutea were obtained from women undergoing surgical sterilization. The tissue was frozen for binding and in situ studies or dispersed for cell cultures. SETTING: Procedures were performed at the San Borja-Arriarán Hospital, National Health Service, and Institute of Maternal and Child Research, Faculty of Medicine, University of Chile. PATIENTS: Twelve patients aged 30 to 40 years requesting surgical sterilization in our institution. The laparotomy was scheduled 6 to 8 days after ovulation. MAIN OUTCOME MEASURES: Expression of IGF-I and IGF-I receptor messenger RNAs (mRNAs) by in situ hybridization. Concentration of IGF-I receptor and binding characteristics. Production of E2 by luteal cells. RESULTS: The binding of IGF-I was detected in middle human CL membranes. In addition, this tissue expressed the mRNAs of IGF-I and its receptor. In culture, IGF-I caused a progressive increase on E2 production. CONCLUSION: These data suggest that the IGF-I system is present in middle human CL. The topographic distribution of IGF-I and its receptors and the ability of IGF-I to stimulate E2 secretion strongly suggest that IGF-I has a role as a paracrine or autocrine regulator of the human luteal function.

Multihormonal regulation of progesterone synthesis in cultured human midluteal cells.

The objectives of this investigation were to examine in vivo insulin like-growth factor-I (IGF-I) secretion by the human midcorpora lutea (mid-CL) and the effects of IGF-I, hCG, FSH, and human GH on progesterone (P) production by CL cells obtained from patients at laparotomy. We first examined whether the CL produces IGF-I by measuring IGF-I levels in the ovarian vein from the ovary bearing the CL. The IGF-I concentration in the ovarian vein bearing the CL (206 +/- 31 ng/mL) was significantly increased compared to the concentration in the contralateral ovarian vein (179.2 +/- 32 ng/mL; P < 0.05). Luteal cells isolated from mid-CL were cultured in serum-free medium 199 in the presence and absence of hCG, FSH, GH, and graded concentrations of IGF-I. At the end of the incubation period (24 h), P levels in the medium were measured by RIA. The treatment with IGF-I (0.1-10 ng/mL) showed a dose-dependent stimulatory action of IGF-I on P synthesis in the luteal cell system, being maximal between 5-10 ng/mL. The treatment with hCG (10 IU/mL), IGF-I (5 ng/mL), and GH (1000 ng/mL) increased basal P synthesis by 300%, 80%, and 30%, respectively (P < 0.001 and P < 0.05). FSH (100 ng/mL), either alone or in combination with IGF-I, failed to stimulate P synthesis. Treatment with IGF-I monoclonal antibody (1:5000) completely reduced P synthesis induced by 5 ng/mL IGF-I and slightly reduced basal P synthesis as well as GH-stimulated P synthesis by human midluteal cells. To further evaluate the specific role of IGF-I on luteal steroidogenesis, IGF-I receptor was identified by chemical cross-linking of [125I]IGF-I to mid-CL membranes. Experiments conducted in the absence and presence of unlabeled IGF-I (500 ng) revealed proteins with characteristics of the type I IGF receptor. These results are consistent with multihormonal regulation of P synthesis by the human mid-CL. hCG and IGF-I play a major role in the stimulation of P synthesis and, to a lesser extent, human GH. These in vivo and in vitro data suggest that the CL is a site of secretion, action, and reception of IGF-I during the midluteal phase.

Progesterone synthesis by human luteal cells: modulation by estradiol.

To assess the role of estradiol (E2) upon progesterone (P4) synthesis, a well defined human midluteal cell system was used. A dose-dependent inhibition of P4 synthesis with and without hCG was induced by E2. In addition, E2 had a dose related cumulative effect on pregnenolone as compared with control experiments (2-fold, P < 0.05) as well as in hCG-stimulated conditions (3-fold, P < 0.005). On the other hand, the concentrations of 20 alpha-hydroxyprogesterone obtained in all experimental conditions were similar to control values, indicating that the catabolism of P4 was not modified. 3 beta-Hydroxysteroid dehydrogenase activity was significantly diminished (P < 0.05) in the presence of E2. Finally, the kinetic studies on P4 synthesis from pregnenolone showed a competitive type of inhibition with a K1 of 2.22 x 10(-6) mol/L. These data indicate an inhibition of 3 beta-hydroxysteroid dehydrogenase on human corpus luteum by E2.

The acrosome reaction-inducing activity of individual human follicular fluid samples is highly variable and is related to the steroid content.

In this study, we have evaluated the relationship between the acrosome reaction-inducing activity of individual human follicular fluid samples and their steroid content. Eighteen samples of follicular fluid were obtained during egg retrieval in six patients undergoing assisted fertilization. Motile spermatozoa were incubated in modified Tyrode's medium (26 mg/ml bovine serum albumin) for 20 h at 1 x 10(7) cells/ml. In a single experiment, aliquots of a semen specimen were simultaneously treated with an aliquot of each follicular fluid sample. The percentage of acrosome reacted spermatozoa was determined using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) lectin. The fluids were also analysed by radioimmunoassay to determine the levels of progesterone, 17 alpha-hydroxy-progesterone, testosterone and oestradiol. The results showed that there was a positive, highly significant correlation between the acrosome reaction-inducing activity and the progesterone level of each follicular fluid sample (r = 0.72, P less than 0.005). Additionally, treatment of the follicular fluid samples with charcoal-dextran caused both a decrease in progesterone concentration and the total loss of the acrosome reaction-inducing activity. The addition of progesterone restored the acrosome reaction-inducing ability in 88% of samples. These data support the idea that progesterone in follicular fluid is the molecule responsible for inducing the acrosome reaction in human spermatozoa.

[Ovulation induction by the chronic administration of naltrexone in a patient with secondary hypothalamic amenorrhea]. / Inducción de ovulación mediante la administración crónica de naltrexona en paciente con amenorrea hipotalámica secundaria.

An ovulatory cycle was induced by oral administration of a specific opiate antagonist: naltrexone, at a dose of 50 mg/day for 26 days in a woman suffering from secondary hypothalamic amenorrhea. The follicular growth was monitored by ultrasound and serial blood measurement of LH, FSH, E2 and progesterone. The hormonal and ultrasound profiles showed an ovulatory cycle with a single dominant follicle. After discontinuation of the treatment, the patient became amenorrheic again. The gonadotropin as well as estradiol plasma levels declined, to that observed before treatment. Naltrexone may be a useful agent for induction of ovulation in women suffering from hypothalamic amenorrhea.

Effect of the aromatase inhibitor 4-hydroxyandrostene-3,17-dione progesterone synthesis by human luteal cells.

The authors studied the effects of 4-hydroxyandrostene-3,17-dione (4-OHA) on progesterone (P), 17 beta-estradiol (E2), and 20 alpha-hydroxy-4-pregnen-3-one synthesis and pregnenolone accumulation in cultured human midluteal cells. A dose-dependent inhibition with and without human chorionic gonadotropin (hCG) of E2 and P production was observed. The accumulation of pregnenolone was significantly enhanced three to fourfold by 4-OHA in this culture system, as compared with control value. In addition, a sevenfold increase on pregnenolone accumulation was observed in the presence of 4-OHA plus 10 IU of hCG as compared with control values and 2.2-fold as compared with the 4-OHA treatments. These in vitro findings indicate a direct effect of 4-OHA on luteal steroidogenesis. Nevertheless, the suppressive effect of 4-OHA on P and E2 production is located at different sites of the steroidogenic pathway. In addition, the results demonstrate that hCG in the presence of 4-OHA stimulated pregnenolone accumulation, suggesting that the inhibition of P synthesis is in some steps after the formation of pregnenolone. These data indicate that the actions of 4-OHA on P or E2 formation have different inhibitory mechanisms.

In vitro net progesterone production by human corpora lutea: effects of human chorionic gonadotropin, dibutyryl adenosine 3',5'-monophosphate, cholera toxin, and forskolin.

Slices of human corpora lutea (CL) obtained at varying stages of the luteal phase from 21 women were used to study the effect of hCG on progesterone (P4) production. Slices obtained from mid- and late CL incubated with 10 IU/mL hCG exhibited a significant increase in net P4 production (P less than 0.001), whereas slices from early CL did not. Mid-CL slices were the most sensitive to hCG (4.2-fold increase in P4 production compared to 1.2-fold for early CL and 2.7-fold for late CL). To investigate the unresponsiveness of early CL to hCG, [125I]hCG binding was studied. All early CL had LH/hCG-specific receptors, and the apparent Kd for this binding was 1.95 X 10(-10) M. Dibutyryl cAMP (1 mM), cholera toxin (0.84 mM), and forskolin (50 microM) stimulated net P4 production (P less than 0.05) in slices of early CL tissue incubated in the presence of methylisobutylxanthine (0.1 mM). Cholera toxin and forskolin stimulated cAMP formation by the early CL, but hCG failed to do so. These results confirm that hCG has an age-dependent stimulatory effect on CL P4 synthesis. Our findings suggest that there is inadequate coupling of the LH/hCG receptor and adenylate cyclase in the early human CL, which explains in part the relative insensitivity of this tissue to the steroidogenic action of hCG.

Immunologic abnormalities in myelofibrosis with activation of the complement system.

Eighteen patients with agnogenic myeloid metaplasia with myelofibrosis were studied for clinical and laboratory evidence of immunologic dysfunction. Clinical findings included the presence of arthritis, vasculitis, and erythema nodosum. Laboratory abnormalities included the presence of circulating immune complexes, antinuclear antibodies, positive direct Coombs tests, elevated latex fixations, and a circulating lupus type anticoagulant. Total hemolytic complement was markedly depressed in four patients. Analysis of complement (C) components C1-C9 and factor B demonstrated significant reduction of only C3 and factor B. By crossed-immunoelectrophoresis, both C3 and factor B, but not C4, were cleaved, indicating that C activation was occurring predominantly via the alternative pathway. The control proteins beta 1H and C3b inactivator were decreased in three of four patients with hypocomplementemia. These data suggest that immunologic mechanisms associated with activation of the complement system play an important role in the disease process of some patients with agnogenic myeloid metaplasia with myelofibrosis.

[Standard and high doses of haloperidol. Clinical, pharmacokinetic and pharmacodynamic aspects]. / Dosis standard y elevada de haloperidol. Aspectos clínicos, farmacocinéticos y farmacodinámicos.

The investigation was designed to study plasma drug concentration and prolactin response determined by two different doses of haloperidol, a standard one and four-fold higher the other, and to explore the relationships between clinical effects and pharmacokinetic and physiological variables in psychotic patients. 24 psychotic patients received haloperidol during 40 consecutive days. During the first 10 days they received 0,25 mg/kg b.w. During the next 20 days they were given 1 mg/kg b.w. Finally they received 0,25 mg/kg b.w. during the last ten days. The patients were clinically assessed and plasma haloperidol and prolactin concentrations were determined on days 0, 5, 10, 20, 30 and 40. In the range of dose studied, the use of higher than conventional doses of haloperidol results in an increased concentration in the cellular site of action and in a more complete blockade of central dopamine receptors. Prolactin response was positive correlated with serum haloperidol concentration. Clinical remission was not direct correlated, neither with serum neuroleptic concentration nor with prolactin level. Those patients who reached higher levels of plasmatic neuroleptic and prolactin concentrations were more susceptible to develop severe extrapyramidal side-effects. Pharmacokinetic and pharmacodynamic monitoring of neuroleptic treatment could be useful to identify these patients.