Rita Canipari

Early programming of maturation competence in mouse oogenesis

Growing mouse oocytes incompetent to mature were freed of attached granulosa cells at different stages of growth, and cultured in vitro in the presence of fibroblast monolayers and/or their products. In these culture conditions, although growth was arrested, isolated oocytes survived in vitro for several days, and finally resumed meiosis spontaneously, progressing up to metaphase I. The culture time length needed for in vitro acquisition of the capacity to mature was inversely related to the initial oocyte size at the time of isolation from granulosa cells, and closely corresponded to developmental timing of acquisition of such ability in vivo. We conclude that the acquisition of mouse oocyte competence to mature follows a definite time program, which is independent of the presence of granulosa cells and of heterologous cell contacts, at least within the developmental stages studied.

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.

TGF-β autocrine loop regulates cell growth and myogenic differentiation in human rhabdomyosarcoma cells

Transforming growth factor β (TGF) is a well‐known inhibitor of myogenic differentiation as well as an autocrine product of rhabdomyosarcoma cells. We studied the role of the TGF‐β autocrine loop in regulating growth and myogenic differentiation in the human rhabdomyosarcoma cell line, RD. We previously reported that the phorbol ester 12‐O‐tetradeca‐noylphorbol‐13‐acetate (TPA) induces growth arrest and myogenic differentiation in these cells, which constitutively express muscle regulatory factors. We show that TPA inhibits the activation of secreted latent TGF‐β, thus decreasing the concentration of active TGF‐β to which the cells are exposed. This event is mediated by the TPA‐induced alteration of the uPA/ PAI serine‐protease system. Complete removal of TGF‐β, mediated by the ectopic expression of a soluble type II TGF‐β receptor dominant negative cDNA, induces growth arrest, but does not trigger differentiation. In contrast, a reduction in the TGF‐β concentration, to a range of 0.14–0.20 × 10−2 ng/ml (which is similar to that measured in TPA‐treated cells), mimics TPA‐induced differentiation. Taken together, these data demonstrate that cell growth and suppression of differentiation in rhabdomyosarcoma cells require overproduction of active TGF‐β; furthermore, they show that a ‘critical’ concentration of TGF‐β is necessary for myogenic differentiation to occur, whereas myogenesis is abolished below and above this concentration. By impairing the TGF‐β autocrine loop, TPA stabilizes the factor concentration within the range compatible for differentiation to occur. In contrast, in human primary muscle cells a much higher concentration of exogenous TGF‐β is required for the differentiation inhibitory effect and TPA inhibits differentiation in these cells probably through a TGF‐β independent mechanism. These data thus clarify the mechanism underlying the multiple roles of TGF‐β in the regulation of both the transformed and differentiated phenotype.—Bouche, M., Canipari, R., Mel‐chionna, R., Willems, D., Senni, M. I., Molinaro, M. TGF‐β autocrine loop regulates cell growth and myo‐genic differentiation in human rhabdomyosarcoma cells. FASEB J. 14, 1147–1158 (2000)

The fungicide mancozeb induces toxic effects on mammalian granulosa cells.

The ethylene-bis-dithiocarbamate mancozeb is a widely used fungicide with low reported toxicity in mammals. In mice, mancozeb induces embryo apoptosis, affects oocyte meiotic spindle morphology and impairs fertilization rate even when used at very low concentrations. We evaluated the toxic effects of mancozeb on the mouse and human ovarian somatic granulosa cells. We examined parameters such as cell morphology, induction of apoptosis, and p53 expression levels. Mouse granulosa cells exposed to mancozeb underwent a time- and dose-dependent modification of their morphology, and acquired the ability to migrate but not to proliferate. The expression level of p53, in terms of mRNA and protein content, decreased significantly in comparison with unexposed cells, but no change in apoptosis was recorded. Toxic effects could be attributed, at least in part, to the presence of ethylenthiourea (ETU), the main mancozeb catabolite, which was found in culture medium. Human granulosa cells also showed dose-dependent morphological changes and reduced p53 expression levels after exposure to mancozeb. Altogether, these results indicate that mancozeb affects the somatic cells of the mammalian ovarian follicles by inducing a premalignant-like status, and that such damage occurs to the same extent in both mouse and human GC. These results further substantiate the concept that mancozeb should be regarded as a reproductive toxicant.

Transforming growth factor-β enhances adhesion of melanoma cells to the endothelium in vitro

Melanoma invasion requires migration through the vascular barrier. An early event in this process is the adhesion of metastatic cells to the endothelium. To elucidate the role of TGF‐β in the regulation of this process, human melanoma SK‐MEL24 cells were labelled with [5′‐3H]‐thymidine and co‐cultured with bovine pulmonary artery endothelial‐cell monolayers. Radioactivity was assumed to be proportional to the number of SK‐MEL24 cells bound to the endothelium. A low number of melanoma cells adhered to endothelial cells in a time‐related manner. Pretreatment for 24 hr with 0.001 to 10 ng/ml TGF‐β1 or TGF‐β2 of both cell types enhanced melanoma‐endothelium adhesion in a dose‐dependent manner. Both melanoma and endothelial cells expressed RI‐ and RII‐type TGF‐β receptors. The effect of TGF‐β was abolished by co‐incubation with the proteoglycan decorin. Conditioned media from melanoma‐endothelium co‐cultures contained latent TGF‐β and failed to affect cell‐cell adhesion. However, activation of TGF‐β by heating the medium or reducing the pH, increased melanoma‐endothelium adhesion to an extent similar to that of the TGF‐β administered to the cultures. Zimography demonstrated that both cell types expressed urokinase‐type plasminogen activator (uPA). Addition of plasminogen to the co‐cultures, which was likely to be activated to plasmin by uPA, resulted in activation of TGF‐β and parallel stimulation of melanoma‐endothelium adhesion. In conclusion, TGF‐β may enhance adhesion of melanoma cells to the endothelium, playing a relevant autocrine/paracrine role in the progression of invasive melanoma. Int. J. Cancer 72:1013–1020, 1997.

Expression localisation and functional activity of pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal polypeptide and their receptors in mouse ovary

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) positively affect several parameters correlated with the ovulatory process. PACAP is transiently expressed in rat preovulatory follicles, while VIP is present in nerve fibres at all stages of development. These two peptides act by interacting with three types of receptors: PACAP type I receptor (PAC1-R), which binds with higher affinity to PACAP, and two VIP receptors (VPAC1-R and VPAC2-R), which bind to PACAP and VIP with equal affinity. The aim of the present study was to characterise the PACAP/VIP/receptor system in the mouse ovary. Results obtained by RT-PCR, immunohistochemistry and in situ hybridisation showed that PACAP was transiently expressed in granulosa cells of preovulatory follicles after human chorionic gonadotrophin (hCG) stimulation, while VIP mRNA was never observed. All the receptors were present in 22-day-old untreated mice. In preovulatory follicles, PAC1-R was expressed both in granulosa cells and in residual ovarian tissue but was stimulated by hCG mainly in granulosa cells; VPAC2-R was present in both the cell compartments and was only mildly stimulated; VPAC1-R was present mainly in the residual ovarian tissue and was downregulated by hCG. PACAP and VIP were equipotent in inhibiting apoptosis in granulosa cells, confirming the presence of functional PACAP/VIP receptors. The contemporary induction by hCG of PACAP and PAC1-R in granulosa cells of preovulatory follicles suggests that, also in mouse ovary, PACAP may play a significant role around the time of ovulation. Moreover, the presence of PACAP/VIP receptors in the untreated ovary suggests a possible role for PACAP and VIP during follicle development.

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.

Characterization, Expression, and Functional Activity of Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors in Human Granulosa-Luteal Cells

CONTEXT Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are found in the ovary of mammalian species, although nothing is known about the possible role of PACAP and VIP in the human ovary. OBJECTIVE We investigated the expression of PACAP and PACAP/VIP receptors in human granulosa-luteal (GL) cells obtained from consenting in vitro fertilization patients attending a private fertility clinic and assessed a possible antiapoptotic effect of these molecules. MAIN OUTCOME MEASURES We measured the expression of PACAP and PACAP/VIP receptor mRNAs in GL cells in response to FSH or LH, as well as the effects of PACAP and VIP on apoptosis. We also evaluated the levels of procaspase-3 in GL cells cultured in the absence of serum. RESULTS After 7 d in culture, GL cells displayed increased responsiveness to FSH and LH (100 ng/ml). FSH and LH promoted PACAP expression, LH doing so in a time-dependent fashion. VIP receptor (VPAC1-R and VPAC2-R) mRNAs were also induced by gonadotropin stimulation. Although PACAP receptor (PAC1-R) mRNA was barely detectable, Western blot analysis revealed its presence. The apoptotic effect of serum withdrawal from the culture environment was reverted by both PACAP and VIP. Both peptides showed the ability to reverse a decrease in procaspase-3 levels induced by culture in the absence of serum. CONCLUSIONS PACAP and VIP appear to play a role in maintenance of follicle viability as a consequence of the antiapoptotic effect. Further studies are warranted to evaluate the respective roles of PACAP and VIP in ovarian physiology and to identify their mechanism of action.

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.

Grape seed extract suppresses MDA-MB231 breast cancer cell migration and invasion.

Background and aimBreast cancer remains a leading cause of mortality among women. In metastasis, cascade migration of cancer cells and invasion of extracellular matrix (ECM) represent critical steps. Urokinase-type plasminogen activator (uPA), as well as metalloproteinases MMP-2 and MMP-9, strongly contribute to ECM remodelling, thus becoming associated with tumour migration and invasion. In addition, the high expression of cytoskeletal (CSK) proteins, as fascin, has been correlated with clinically aggressive metastatic tumours, and CSK proteins are thought to affect the migration of cancer cells. Consumption of fruits and vegetables, characterized by high procyanidin content, has been associated to a reduced mortality for breast cancer. Therefore, we investigated the biological effect of grape seed extract (GSE) on the highly metastatic MDA-MB231 breast cancer cell line, focusing on studying GSE ability in inhibiting two main metastatic processes, i.e., cell migration and invasion.MethodsAfter MDA-MB231 breast cancer cells stimulated with GSE migration and invasion were evaluated by means of trans-well assays and uPA as well as MMPs activity was detected by gelatin zymography. Fascin, β-catenin and nuclear factor-κB (NF-κB) expression were determined using western blot technique. β-Catenin localization was observed by confocal microscopy.ResultsWe observed that high concentrations of GSE inhibited cell proliferation and apoptosis. Conversely, low GSE concentration decreased cell migration and invasion, likely by hampering β-catenin expression and localization, fascin and NF-κB expression, as well as by decreasing the activity of uPA, MMP-2 and MMP-9.ConclusionsThese results make GSE a powerful candidate for developing preventive agents against cancer metastasis.