Wide bandwidth fiber-optic ultrasound probe in MOMS technology: Preliminary signal processing results.

An ultrasonic probe consisting of two optical fiber-based miniaturized transducers for wideband ultrasound emission and detection is employed for the characterization of in vitro biological tissues. In the probe, ultrasound generation is obtained by thermoelastic emission from patterned carbon films in Micro-Opto-Mechanical-System (MOMS) devices mounted on the tip of an optical fiber, whereas acousto-optical detection is performed in a similar way by a miniaturized polymeric interferometer. The microprobe presents a wide, flat bandwidth that is a very attractive feature for ultrasonic investigation, especially for tissue characterization. Thanks to the very high ultrasonic frequencies obtained, the probe is able to reveal different details of the object under investigation by analyzing the ultrasonic signal within different frequencies ranges, as shown by specific experiments performed on a patterned cornstarch flour sample in vitro. This is confirmed by measurements executed to determine the lateral resolution of the microprobe at different frequencies of about 70µm at 120MHz. Moreover, measurements performed with the wideband probe in pulsed-echo mode on a histological finding of porcine kidney are presented, on which two different spectral signal processing algorithms are applied. After processing, the ultrasonic spectral features show a peculiar spatial distribution on the sample, which is expected to depend on different ultrasonic backscattering properties of the analyzed tissues.

Effects of hypoxia on endothelin-1 sensitivity in the corpus cavernosum.

The penis remains in a hypo-oxygenated, flaccid state for a large majority of the time. In this study, we investigated the effect of changing oxygen tension on the expression and functional activity of endothelin-1 (ET-1) receptors in the penis. Experiments were performed in rabbit and human corpora cavernosa (CC) as well as in human fetal penile tissue and cell cultures [human fetal penile endothelial cells (hfPECs) and human fetal smooth muscle cells (hfPSMCs)]. Endothelin A (ETA) receptors are expressed by both endothelial and muscular cells in all tissues investigated. Only penile endothelial cells express endothelin B (ETB) receptors, which are further turned on during experimental hypoxia. In addition, hypoxia also allows ETB expression in the muscular compartment without affecting ETA expression. This hypoxia-induced over-expression of ETB decreased the contractile activity of ET-1 and increased ETB-mediated relaxation. The latter was essentially related to increased ETB-mediated nitric oxide formation in hfPEC and even in hfPSMC. Hypoxia also induced a time-dependent down-regulation of RhoA and Rho kinase (ROK) expression which, in turn, participated in the decreased contractile activity of ET-1 in the hypoxic penile tissue. Accordingly, during hypoxia, an ROK inhibitor, Y27632, was less effective in relaxing ET-1-precontracted strips. In conclusion, prolonged (24 h) hypoxia stimulated several counter-regulatory mechanisms in penile tissue, including up-regulation of ETB and down-regulation of RhoA/ROK pathways, which may help to preserve CC hypo-oxygenation, allowing smooth muscle relaxation and, most probably, penile erection.

Expression of functional estrogen receptors in human fetal male external genitalia.

It is generally assumed that male genital development is determined by androgens on a default program leading to female genitalia. Female genitalia virilization is due to high levels of androgens, whereas feminization is linked to reduction or lack of fetal androgen. Excess androgen determines sex reversion in female, whereas excess estrogen does not cause male feminization. In the present study, we investigate the presence of androgen receptors (AR) and estrogen receptors (ER) in human fetal penile tissue and in a cellular model of human fetal penile smooth muscle cells (hfPSMC). By immunohistochemistry, we showed the presence of ER and AR in the developing penile tissue of male fetuses. Besides the presence of AR, hfPSMC showed ERalpha/beta as demonstrated by RT-PCR, Western blot, and binding techniques. These receptors are functionally active because cell stimulation with 17beta-estradiol increased progesterone receptor B expression and inhibited hfPSMC growth, both effects being reversed by tamoxifen. Conversely, cell proliferation was stimulated by R1881 and testosterone, an effect enhanced by letrozole. These findings are the first demonstration of the presence of functional ER in differentiating male external genitalia and indicate a possible novel inhibitory role of estrogens in the regulation of the development of these sex structures.

Expression and regulation of endothelin-1 and its receptors in human penile smooth muscle cells.

We report for the first time that penile smooth muscle cells (SMC) not only respond to, but also synthesize, endothelin-1 (ET-1), one of the main regulators of SMC activity. Immunohistochemical studies indicated that, beside endothelial cells (EC), SMC of the human adult and fetal penis also express ET-1 and its converting enzyme, ECE-1. Accordingly, cultures of adult penile stromal cells express these genes. We also prepared and characterized penile SMC from human fetuses. These cells express SMC specific markers such as alpha smooth muscle actin and phosphodiesterase type 5A3 along with hallmarks of androgen-dependent cells (androgen receptor and 5alpha reductase type 2). Human fetal penile SMC (hfPSMC) are immunopositive for ET-1 and release ET-1. ET-1 expression in hfPSMC was strongly increased by several factors such as transforming growth factor-beta1 (TGF-beta1), interleukin-1alpha (IL-1alpha), ET-1 itself and prolonged (24 h) hypoxia. This latter condition not only affected ET-1 expression but also responsiveness. While at normal oxygen tension, hfPSMC responded to ET-1 with a decreased proliferation mediated by the endothelin-A receptors and TGF-beta1; however, during hypoxia, ET-1 stimulated cell growth. Accordingly, prolonged hypoxia up-regulated endothelin-B receptor mRNA expression. In conclusion, our results indicate that in penile tissues SMC produce ET-1 and that such production is modulated by factors involved in penile physiology and tissue remodelling. In addition, the hfPSMC we have characterized might be a useful model for studying biochemical aspects of the human erectile process in vitro.

Comments on "Frequency decomposition and computing of ultrasound medical images with wavelet packets".

In this paper, errors and discrepancies in the subject paper [Cincotti et al., (2002)] are highlighted. A comment, concerning the axial resolution associated to the adopted processing procedure is also reported.

Endothelium-dependency of yohimbine-induced corpus cavernosum relaxation.

Development and maintenance of penile erection requires the relaxation of the smooth muscle cells in the cavernous bodies and is essentially mediated by nitric oxide (NO). The penile flaccid state is conversely maintained by the alpha adrenergic neuroeffector system and by other vasoconstrictors, such as endothelin-1 (ET-1). In this study we examined the mechanisms involved in yohimbine-induced relaxation in human and rabbit corpora cavernosa (CC). We essentially found that yohimbine not only blocks contractions induced by adrenergic agonists, but also by non-adrenergic substances, such as ET-1. This effect was unrelated to antagonism at the level of ET receptors, because yohimbine did not affect ET-1-induced increase in intracellular calcium in isolated CC cells. Conversely, our data suggest that yohimbine counteracts ET-1-induced contractions by interfering with NO release from the endothelium. In fact, yohimbine-induced CC relaxation was inhibited by the mechanical removing of the endothelium and by blocking NO formation or signalling via guanylate cyclase and cGMP formation. Conversely, yohimbine activity was strongly increased by inhibiting cGMP degradation. In an experimental model of hypogonadism, performed on rabbits by chronic treatment with a long-lasting GnRH agonist, the relaxant yohimbine activity was also decreased, but completely restored by androgen supplementation. This effect was evident only in preparations in which the main source of NO was present (endothelium) or in which NO formation was not impaired by L-NAME. Our data indicate that the relaxant effect of yohimbine is both endothelium and androgen-dependent. This might justify the lack of efficacy of this drug in treatment of some form of organic erectile dysfunction.

Identification, localization and functional activity of oxytocin receptors in epididymis.

Oxytocin (OT) is a neurohypophysial hormone with unclear physiological functions in the male. Several previous studies indicated that OT might have a role in the ejaculatory process, stimulating sperm release from the epididymal storage. In this study we investigated on the presence and function of OT receptor (OTR) in rabbit and human epididymis. By using RT-PCR, Western and binding studies, we found that OTR gene and protein is expressed in the human epididymis and stimulates in vitro contractility. The immunolocalization of OTR suggests that the receptor is not only present in the smooth muscle cells of the human epididymis but also in the epithelial compartment. Experiments performed in rabbit epididymal epithelial (rEE) cells in culture indicate that OT induces the release of an other potent stimulator of epididymal contractility, endothelin-1 (ET-1), Blocking the ET(A) subtype of the ET-1 receptors, by using a specific antagonist (BQ-123), partially counteracts the contractile effect of OT, suggesting positive interactions between the two peptides in regulating epididymal contractility. Finally, to investigate whether an acute OT administration increases sperm release also in humans, we treated oligozoospermic patients with an intravenous bolus of OT (2.5 IU), just before sperm collection. In a small, single blind study, we found that OT almost doubled sperm retrieval when compared with vehicle administration. Our results indicate that OT might have physiological functions also in the male, controlling epididymal motility and sperm progression through the male genital tract.

Endothelin-1 production by prostate cancer cell lines is up-regulated by factors involved in cancer progression and down-regulated by androgens.

BACKGROUND: Recent data demonstrate that endothelin-1 (ET-1) concentration increases in plasma of men with advanced, hormone-refractory prostate adenocarcinoma. In addition, ET-1 is involved in osteblastic remodelling and new bone formation, suggesting a role for this vasoactive peptide in the metastatic progression of prostate cancer to the bone. METHODS: We investigated the regulation of ET-1 expression in androgen-sensitive and insensitive prostate cancer cell lines by androgens and several factors involved in progression of prostate cancer (EGF) and bone remodelling (TGFbeta-1, IL1-alpha and IGF-1). RESULTS: Northern analysis and radio immunoassay demonstrated that all the ET-1 pathways are tuned off in the androgen-sensitive LNCaP cell line when compared to the androgen-insensitive PC-3 and DU145. In PC-3 cells transfected with a full-length androgen receptor expression vector (PC-3-AR), treatment with androgens reduced gene expression and secretion of ET-1 without affecting the gene expression of ET-3. Collectively, these data support a role for androgens in the regulation of ET-1 production by prostate adenocarcinoma cells. In PC-3 and DU145 cells, ET-1 gene expression and secretion were up-regulated by TGFbeta-1, EGF and IL1-alpha, whereas IGF-1 was ineffective. Conversely, none of the treatments affected ECE-1 or ET-3 gene expression. CONCLUSIONS: In conclusion, ET-1 production by prostate adenocarcinoma cells is down-regulated by androgens and up-regulated by factors involved in tumour progression indicating a role for this peptide in the biology of prostate cancer. In view of the role exerted by ET-1 in the process of bone metastasis, our data suggest the use of ET-1 receptor antagonists in the treatment of advanced prostate cancer.

Uteroglobin reverts the transformed phenotype in the endometrial adenocarcinoma cell line HEC-1A by disrupting the metabolic pathways generating platelet-activating factor.

Uteroglobin, originally named blastokinin, is a protein synthesized and secreted by most epithelia, including the endometrium. Uteroglobin has strong anti-inflammatory properties that appear to be due, at least in part, to its inhibitory effect on the activity of the enzyme phospholipase A(2). In addition, recent experimental evidence indicates that uteroglobin exerts antiproliferative and antimetastatic effects in different cancer cells via a membrane receptor. The human endometrial adenocarcinoma cell line HEC-1A does not express uteroglobin. Thus, we transfected HEC-1A cells with human uteroglobin cDNA. The transfectants showed a markedly reduced proliferative potential as assessed by impaired plating efficiency as well as by reduced growth in soft agar. Cytofluorimetric analysis clearly indicated that in uteroglobin-transfected cells the time for completion of the cell cycle was increased. We previously demonstrated that HEC-1A cells actively synthesize platelet-activating factor, one of the products of phospholipase A(2) activity. In addition, we demonstrated that platelet-activating factor stimulates the proliferation of these cells through an autocrine loop. In uteroglobin transfectants, the activity of phospholipase A(2) and platelet-activating factor acetyl-transferase, which are involved in the synthesis of platelet-activating factor, was significantly reduced compared with wild-type and vector-transfected cells (p < 0.05). Our results indicate that enforced expression of uteroglobin in HEC-1A cells markedly reduced their growth potential and significantly impaired the synthesis of platelet-activating factor, an autocrine growth factor for these cells. These data suggest that one possible mechanism for the recently observed antineoplastic properties of uteroglobin may be the inhibition of the synthesis of platelet-activating factor.

Sex steroids and odorants modulate gonadotropin-releasing hormone secretion in primary cultures of human olfactory cells.

Olfactory neurons and GnRH neurons share a common origin during development. In the nasal epithelia, GnRH neurons persist throughout fetal life and adulthood. The fate and function of these neurons in vivo have remained unknown. In a previous in vitro study, we isolated, cloned, and propagated primary long term cell cultures from the olfactory neuroepithelium of 8- to 12-week-old human fetuses. These cells expressed both neural proteins as well as olfactory genes and were responsive to odorant stimuli. We now report that these human olfactory cells also express the GnRH gene and protein. Combined HPLC and RIA studies have indicated that these cells release authentic GnRH in spent media. The release of GnRH was time dependent and was positively affected by sex steroids and odorants. Immunohistochemical data demonstrated the presence of sex steroid receptors in these cells. The presence of the alpha- and beta-subtypes of the estrogen receptor was also demonstrated by RT-PCR and Western blot analysis. When the cells were stimulated with increasing concentrations of 17beta-estradiol in the presence of a fixed concentration of progesterone (10(-7) mol/L), the combination of the two steroids induced a 3- to 4-fold increase in GnRH secretion. This stimulatory effect was completely blunted by tamoxifen. Neither 17beta-estradiol nor progesterone was effective when tested separately. Treatment with increasing concentrations of the odorant, l-carvone, induced a time- and dose-dependent dramatic increase in GnRH protein release (1000-fold increase) and gene expression. Repeated application of the stimulus resulted in a progressive lower responsiveness of the cells. To our knowledge, this is the first time that primary cell cultures from human fetal olfactory neuroepithelium have been shown to express and release GnRH. Our results also demonstrate that these cultures, which are sensitive to sex steroids and odorants, can be useful models in the study of the complex array of regulatory factors that finely tune GnRH secretion in humans.

Screening for microdeletions of Y chromosome genes in patients undergoing intracytoplasmic sperm injection.

The potential of assisted reproduction techniques to transmit genetic defects causing male infertility raises questions concerning the need for a systematic genetic screen and counselling. Deletions of the long arm of the Y chromosome are frequently associated with a failure of spermatogenesis. The search for Y specific sequences and for the gene families RNA binding motif (RBM) and deleted in azoospermia (DAZ) have been introduced in many laboratories. The incidence of Y microdeletions varies widely between studies, from 1-55%. These differences are mainly related to study design. The highest incidence of microdeletions has been reported in well selected idiopathic azoospermic patients. Since microdeletions have been reported also in non-idiopathic patients, it is important to define what is the deletion frequency in unselected patients. We report Y chromosome microdeletion screening in 134 unselected patients undergoing intracytoplasmic sperm injection (ICSI). In the first part of the study we tested six Y chromosome markers. We found three patients with microdeletions (2.2%). Subdivision of the study population revealed a deletion incidence of 4.7% in azoospermic/cryptozoospermic patients; an incidence of 7% in idiopathic patients and an incidence of 16% in idiopathic azoospermic/cryptozoospermic patients. The second part of the study consisted of a screen for the presence of the Y chromosome genes, DBY, CDY, XKRY, eIF-1A, DAZ and BPY2. No additional gene-specific deletions were found. Further data on gene specific screening are needed especially for selected idiopathic patients.

Cyclic adenosine 3',5'-monophosphate-responsive element modulator gene expression in germ cells of normo- and oligoazoospermic men.

In about one third of infertile men the cause of impaired spermatogenesis is not known. Spermatogenesis appears to be mediated at least in part by the pituitary gonadotropins, which activate the cAMP-dependent signaling pathway. The end point of this pathway is the activation of nuclear transcription factors, such as cAMP-responsive element-binding protein and cAMP-responsive element modulator (CREM). These factors, upon binding to gene sequences identified as cAMP response elements, modulate the expression of germ cell-specific genes that, in turn, promote the completion of spermatogenesis. The expressions of the cAMP-responsive element-binding protein and CREM genes create different isoforms, which can be divided into two groups: activators or repressors of gene regulation. Only CREM repressors are expressed in premeiotic germ cells in mice, whereas a switch to the expression of the CREM activator tau is observed from postmeiotic germ cells onward. Completion of germ cell maturation appears to be dependent on this phenomenon. Recently, mice lacking CREM gene expression have been generated. These animals were infertile and presented a developmental arrest of germ cell maturation at the stage of early spermatid. In this report we demonstrate that CREM gene expression also occurs in human germ cells. In particular, we determined by RT-PCR that a switch from the expression of CREM repressors to CREM activators is present in postmeiotic germ cells in normospermic men. Conversely, in oligoazoospermic patients only the expression of CREM repressors was detected. These data were confirmed by in situ hybridization studies in which transcripts for CREM activators were detected in postmeiotic germ cells in testis specimens showing conserved spermatogenesis, but not in specimens showing maturation arrest at the spermatid stage. Thus, our results indicate that the lack of a switch in the expression of CREM gene isoforms may be related to impaired spermatogenesis in humans.

Endothelin-1 is synthesized and biologically active in human epididymis via a paracrine mode of action.

In a previous study, we reported the presence of endothelin-1 and endothelin receptors in the human testis. We have now extended our investigations to the human epididymis. Since sperm appear to be immotile during their transit through the epididymis, it is conceivable that specific local factors promote smooth muscle contraction, enabling sperm transport. In this paper, we show that endothelin-1 mRNA and protein are readily detectable in the epithelial compartment of the human epididymis, and that endothelin converting enzyme- 1, which converts the precursor pro-endothelin-1 into active endothelin-1, is expressed in the epididymis, consistent with active processing of the prohormone. In addition, two classes of endothelin receptors were characterized and located in the muscle cells of the epididymis. These receptors correspond, in terms of affinity constants and capacity, to the previously characterized endothelinA and endothelinB receptor. These receptors appear to be biologically active and mediate the contractile activity of the epididymis in vitro. Our data suggest that endothelin-1, via a paracrine mode of action, may be responsible for sperm progression through this organ.

Gene expression of endothelin-1, endothelin-converting enzyme-1, and endothelin receptors in human epididymis.

We have previously reported the presence of endothelin-1 (ET-1) and its receptors in the human testis. In the present study we extended our investigations to human epididymis. The rationale of our study originated from the fact that sperm appear to be immotile during their transit through the epididymis. Hence, it is conceivable that specific factors, unknown to date, are present in this organ, capable of inducing smooth muscle contractions, thus forcing sperm transport. In this paper it is shown that ET-1 messenger ribonucleic acid and protein are readily detectable in the epithelial compartment of the human epididymis, and that ET-converting enzyme-1, which converts the precursor pro-ET-1 into the active peptide ET-1, is expressed in the epididymis, thus indicating an active processing of the prohormone. In addition, two classes of ET receptors were characterized and located in the muscle cells of the epididymis. These receptors correspond, in terms of affinity constants and capacity, to the ETA and ETB receptors previously characterized. These receptors mediate the contractile activity of the epididymis in vitro, thus suggesting that ET-1 can be responsible of sperm progression through this organ, acting via a paracrine mode of action.

Interferon-alpha downregulates expression of the oxytocin receptor in cultured human myometrial cells.

Previous studies in the endometrium of ruminants showed that type I interferon (IFN) prevents oxytocin receptor (OTR) formation. We studied the effect of IFN-alpha on human myometrial cells in culture expressing a high density of biologically active OTR. We found that IFN-alpha induced a 35-50% decrease in OTR mRNA and protein and that this inhibition was time and dose dependent. Maximal inhibition of OTR mRNA was obtained after 2-3 days, whereas 1-(beta-mercapto-beta, beta-cyclopentamethyl-enepropionic acid,2-O-Me-Tyr,Thr4,Orn8,Tyr9-amide)-[125I]vasotocin ([125I]OTA) binding reached a nadir after 3-4 days, with half-maximal inhibitory concentration (IC50) = 1,100 U/ml. Mathematical analysis of multiple homologous competition curves for [125I]OTA indicated that IFN-alpha treatment (5,000 U/ml x 3 days) reduced just the binding capacity (Bmax) without changing the binding affinity. Accordingly, the same treatment with IFN-alpha did not affect the half-maximally effective concentration (EC50) for the oxytocin-induced increase in intracellular calcium but significantly decreased maximal responsiveness (Emax) of myometrial cells to OT stimulation. In conclusion, our data demonstrate, for the first time, a negative regulation by IFN-alpha of the steady-state expression of OTR mRNA in cultured human myometrial cells obtained from nonpregnant uteri. This inhibition was followed by a parallel decrease in both the Bmax for [125I]OTA and Emax for oxytocin, suggesting a decreased OTR protein availability.

Quantitation of somatostatin receptor type 2 gene expression in neuroblastoma cell lines and primary tumors using competitive reverse transcription-polymerase chain reaction.

We previously reported the presence of somatostatin (SS-14)-binding sites in a wide panel of human neuroblastoma (NB) tumor cell lines. Given that the adrenal gland and its relative embryonal and adult tumors express an abundance of mRNA for somatostatin receptor type 2 (sst2) mRNA, we studied the quantitative expression of sst2 in 6 NB cell lines and 15 primary tumors using competitive reverse transcription (RT)-PCR. This method uses an insertion mutant of the target gene as a competitor for the RT-PCR reaction, thus allowing exact quantitation of sst2 mRNA abundance. We found expression of specific transcripts for sst2 in all of the NB cell lines and tumors investigated (range, 9 x 10(5)-4 x 10(9) molecules/microg RNA). In NB cells, the expression of sst2 was highly correlated with SS-14-binding sites (R = 0.93). In primary tumors, sst2 was positively related to the expression of the neuroendocrine marker secretogranin II (P < 0.05) and negatively related to N-myc amplification (a poor prognostic factor, P < 0.005) and metastatic dissemination (P < 0.05). In addition, Kaplan-Meier curves indicate that sst2 expression is positively related to survival (P = 0.01). In a patient with stage IVs disease (a spontaneously regressing form), we found the highest sst2 expression (4 x 10(9) molecules/microgram RNA), a value relatively similar to that of normal adrenal. In conclusion, these data indicate that quantitation of sst2, as assessed with competitive RT-PCR, could represent a new prognostic tool in the neuroendocrine tumor NB. Since sst2 recognizes octreotide with high affinity, these findings could also have both diagnostic and therapeutic value.

Expression of endothelin-1 gene and protein in human granulosa cells.

Previous studies in animal models indicated an autocrine/paracrine action of endothelin-1 (ET-1) in the ovary. We now report evidence on the presence of ET-1 in human ovary during reproductive life. Immunohistochemical and in situ hybridization studies demonstrated a positive signal into cytoplasm of granulosa cells (GC) of follicles at different growth stages. The concentration of ET-1-like immunoreactivity (ET-1-LI) was also measured by a specific RIA in human follicular fluid (FF). FF samples were obtained from women in an in vitro fertilization program undergoing gonadotropin stimulation (group A; n = 24) or no treatment (group B; n = 7). The mean (+/-SD) ET-1-LI FF level in group A (4.85 +/- 2.06 pg/mL) was significantly higher than that in group B (1.29 +/- 0.43 pg/mL; P < 0.01), whereas the corresponding mean plasma levels were not significantly different and were not correlated to respective FF values. Our results indicate for the first time the presence of ET-1 and its messenger ribonucleic acid in the GC of the human ovary. The higher ET-1-LI levels found in the FF from women undergoing gonadotropin treatment suggest a modulation by gonadotropins and/or ovarian steroids of ET-1 production by GC.