Fiorella Miceli

Stromal-epithelial interactions modulate estrogen responsiveness in normal human endometrium

Abstract The coculture of endometrial epithelial cells (EEC) with stromal cells (ESC) allows achievement of an improved in vitro system for studying interactions between cells via soluble signals. The purpose of this study was to investigate whether 17β-estradiol and insulin can induce proliferation of EEC through ESC-secreted factors. No evidence of estrogen-induced EEC proliferation has been reported so far in the conventional culture methods. To this end, we used an in vitro bicameral coculture model where human EEC were grown on extracellular matrix-coated inserts applied in dishes containing ESC. Proliferation was assessed by tritiated thymidine incorporation. Homogeneity of endometrial cell populations was ascertained immunocytochemically. 17β-Estradiol did not induce any proliferative effect on EEC cultured alone. Endometrial epithelial cell proliferation was significantly enhanced in EEC/ESC cocultures; moreover, it was further increased by 17β-estradiol addition. Insulin increased proliferation in EEC cultured alone, but again the effect was more pronounced in EEC/ESC cocultures. Coincubation of 17β-estradiol and an antibody against insulin-like growth factor I (IGF I) led to neutralization of ESC-mediated EEC proliferation. This work provides evidence that the effect of 17β-estradiol on human EEC proliferation may be mediated at least in part through ESC-secreted IGF I. We also showed that insulin effect is also partially due to ESC activation.

Growth hormone induces in vitro maturation of follicle- and cumulus-enclosed rat oocytes

The aim of this study was to assess the possible role of growth hormone (GH) on rat oocyte maturation. This effect was analyzed in follicle-enclosed, cumulus-enclosed and denuded oocytes obtained from immature pregnant mares serum gonadotropin (PMSG)-treated rats. The addition of GH to the cultures significantly accelerated maturation in both follicle- and cumulus-enclosed oocytes while no effect was seen on denuded oocytes maturation. Also, GH accelerated meiotic maturation in follicle-enclosed oocytes from immature untreated rats. The GH action was not mediated by lactogenic receptors since prolactin (Prl) did not affect the maturation process while it was mediated by insulin growth factor-I (IGF-I) as suggested by the block of GH action observed in the presence of antibodies anti-IGF-I. Finally, no GH effect was found when dbcAMP was added to the cultures. Our results demonstrate that GH is capable of inducing maturation in oocytes from both primed and unprimed rats. Since the presence of physiological levels of GH in the ovary is now well established, the present data strongly suggest a potential relevance of GH in the reproductive biology.

Growth hormone induction of rat granulosa cell tissue-plasminogen activator expression and progesterone synthesis

The plasminogen activator (PA) system is present in the ovary and appears to be involved both in follicular growth and ovulation. Similarly, the growth hormone (GH) has been demonstrated to positively affect some ovarian activities. Interestingly, GH appears not only as a mediator of gonadotropin effects, but also as having an independent action of its own on the ovary. In the present study we wanted to investigate if GH could affect ovarian plasminogen activator (PA) activity and steroidogenesis. Granulosa cells from immature rats, injected with pregnant mare serum gonadotropin (PMSG) for inducing follicular growth, were cultured for 24 h with increasing concentrations of GH. A significant dose-dependent increase in tPA activity was observed in the GH-treated cells. This effect was exerted at the mRNA level and the use of cycloheximide, a protein synthesis inhibitor, suggested that GH did not require any other intermediary protein for inducing tPA-mRNA. Furthermore, cAMP levels were not affected by GH treatment. Finally, GH was found to increase progesterone (P) synthesis by granulosa cells. The correlation between the PA system and ovulation and the importance of a normal steroidogenesis for the ovarian physiology claim for a key role of GH in the ovarian activities.

Effects of Nicotine on Human Luteal Cells In Vitro: A Possible Role on Reproductive Outcome for Smoking Women

Abstract We investigated the effect of nicotine and its methylated metabolite, N-methyl-nicotine (M-nicotine), on human luteal cells by measuring release of progesterone and prostaglandins (PGs) from cultured cells and by testing gene expression of vascular endothelial growth factor (VEGF), an angiogenic factor strictly involved in luteal pathophysiology. Primary cultures of human luteal cells were treated for 24 h with nicotine and M-nicotine (from 10−6 to 10−11 M) either alone or combined with hCG (25 ng/ml); progesterone and PGs were assayed in the culture medium. In another group of experiments, luteal cells were treated for 24 h with nicotine and M-nicotine (10−7 M) to perform reverse transcriptase-polymerase chain reaction on VEGF mRNA. Nicotine and M-nicotine negatively affected basal luteal steroidogenesis at all tested concentrations, but neither was able to affect hCG-induced progesterone release. Both substances were able to significantly increase PGF2α release from luteal cells, with a dose-related efficacy for M-nicotine. On the contrary, PGE2 release was significantly inhibited by both nicotine and its metabolite. Finally, nicotine was able to increase VEGF mRNA expression significantly, whereas M-nicotine was not. In conclusion, nicotine and M-nicotine can induce a sort of luteal insufficiency by inhibiting progesterone release, probably through modulation of the PG system.

Control of human luteal steroidogenesis: role of growth hormone-releasing hormone, vasoactive intestinal peptide, and pituitary adenylate cyclase-activating peptide

OBJECTIVE To examine the possible effect of growth hormone-releasing hormone (GHRH), vasoactive intestinal peptide, and pituitary adenylate cyclase-activating peptide on basal and hCG-stimulated P production by human luteal cells. DESIGN Cultures of human luteal cells from the early and midluteal phase. SETTING All corpora lutea were obtained from the Obstetrics and Gynecology Department of the Università Cattolica, a public care center. PATIENT(S) Ten nonpregnant women between 35 and 47 years of age underwent surgery for various nonendocrine disorders, such as leiomyomatosis. INTERVENTION(S) Corpora lutea were obtained at the time of hysterectomy. MAIN OUTCOME MEASURE(S) Luteal cells were incubated with GHRH, vasoactive intestinal peptide, and pituitary adenylate cyclase-activating peptide with or without hCG at different concentrations. RESULT(S) Pituitary adenylate cyclase-activating peptide stimulated P production in a dose- and time-dependent manner, whereas GHRH and vasoactive intestinal peptide did not affect luteal steroidogenesis. None of the three peptides were found to synergize with hCG. CONCLUSION(S) Pituitary adenylate cyclase-activating peptide can influence human luteal steroidogenesis.

Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Plasminogen Activator Expression in Rat Granulosa Cell

Abstract Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide isolated from ovine hypothalamus. It has been demonstrated to be transiently expressed in preovulatory follicles and to positively affect several parameters correlated with the ovulatory process. The aim of the present study was to investigate whether PACAP influences the plasminogen/plasmin system in rat ovary. Plasminogen activators (PAs) are serine proteases, modulated by gonadotropins and several peptides in preovulatory follicles, that appear to be involved in ovulation. Granulosa cells obtained from immature eCG-treated rats were cultured for 24 h in the presence of increasing concentrations of PACAP and vasoactive intestinal peptide (VIP). A significant, dose-dependent increase in tissue-type PA (tPA) activity and decrease in urokinase-type (uPA) PA activity were observed in PACAP-treated cells. These effects were exerted at the mRNA level. The use of cycloheximide, a protein synthesis inhibitor, suggested that PACAP requires an intermediary protein to decrease uPA-mRNA, but not to induce tPA-mRNA. However, no significant modulation of PAs was observed in the presence of VIP. When granulosa cells were stimulated within the intact follicle (i.e., maintaining the three-dimensional structure and in the presence of the theca cell layers), both PACAP and VIP dose-dependently stimulated tPA. These data suggest that, in addition to the PACAP type I receptor present on granulosa cells, different subtypes of PACAP receptors are present in the different ovarian compartments.

Growth hormone stimulates androsterone synthesis by rat theca-interstitial cells

The aim of this study was to assess the possible role of growth hormone (GH) on androsterone synthesis. This effect was analyzed in theca-interstitial cells obtained from immature female rats. The addition of GH to the cultures significantly stimulated androsterone (A) synthesis in a dose- and time-dependent way and this effect was not due to a cellular number increase. When added to the hCG cultures, GH significantly enhanced androgen production even though it did not synergyze with the chorionic gonadotropin. The addition of antibodies anti-IGF-I to the GH cultures did not modify the growth hormone effect suggesting that GH probably does not require IGF-I to achieve its effect on A production. Finally, no effect of GH on cAMP levels were observed in the cultures at the end of the treatment. Our results demonstrate that GH is able to significantly induce A synthesis by rat theca-interstitial cells. Since the presence of GH and its receptors in the ovary is now well established the present data strongly suggest a potential relevance of GH in reproductive biology.

Insulin-like growth factor (IGF)-I and IGF-II stimulate progesterone production by human luteal cells: role of IGF-I as mediator of growth hormone action

OBJECTIVE To examine the possible direct effect of insulin-like growth factor (IGF)-I and IGF-II on basal and hCG-stimulated P production by cultured human luteal cells. The possible role of IGF-I as mediator of GH action on luteal steroidogenesis also was investigated. DESIGN Cultures of human luteal cells from early and midluteal phase. SETTING All corpora lutea were obtained from the Obstetrics and Gynecology Department of the Universita Cattolica, a public care center. PATIENTS Eight nonpregnant women between 35 and 47 years of age underwent surgery for various nonendocrine disorders such as leiomyomatosis. INTERVENTIONS Corpora lutea were obtained at the time of hysterectomy. MAIN OUTCOME MEASURES Luteal cells were incubated with IGF-I or IGF-II with or without hCG at different concentrations. Growth hormone also was used alone and with an anti-IGF-I-antibody. RESULTS We found that IGF-I and IGF-II were able to stimulate directly the P production at all used concentrations and that both of them significantly amplified the steroidogenic hCG effect. Finally, IGF-I was shown to mediate the positive GH action on P synthesis.

Endothelin-1 : Expression and role in human corpus luteum

PROBLEM: Several recent data suggest an involvement of endothelin (ET)‐1, a powerful vasoconstrictor peptide, in reproductive function. This study was designed to investigate the presence and role of ET‐1 in human corpus luteum.

Growth hormone-releasing factor stimulates meiotic maturation in follicle- and cumulus-enclosed rat oocyte

This study was designed in order to assess the possible role of growth hormone-releasing factor (GRF) on oocyte maturation. This effect was analyzed in follicle-enclosed, cumulus-enclosed and denuded oocytes obtained from immature pregnant mares serum gonadotropin-treated rats. The addition of GRF to the cultures significantly accelerated maturation in follicle- and cumulus-enclosed oocytes while no effect was seen on denuded oocytes. Also, the neuropeptide was able to induce maturation in follicle-enclosed oocytes obtained from immature untreated rats. The GRF action was probably not mediated by the vasoactive intestinal peptide (VIP) receptors since the two hormones had different effects on oocyte maturation and on cAMP production by granulosa cells. In addition the disappearance of the GRF effect observed in the presence of antibodies anti-GH suggested that GRF required the intermediacy of GH to accomplish its effect on oocyte maturation. Finally, GRF did not affect meiotic maturation when dbcAMP was added to the cultures. Our results demonstrate the ability of GRF to accelerate maturation in oocytes from both primed and unprimed rats. Since the presence and the involvement of GRF at the ovarian levels is now well established, the present data strongly suggest an important potential role of GRF in the ovarian physiology.