By examining in vitro the effects of prostaglandin E-2 (PGE-2) and prostaglandin F-2 (PGF-2 ) induced in the corpora lutea (CL) of pseudopregnant rabbits, we have demonstrated that these prostaglandins modulate luteal nitric oxide synthase (NOS) activity and progesterone production differently, depending on the age of the CL. On CL obtained on day 4 of pseudopregnancy (day-4), PGE-2 was found to depress NOS total activity to 13% of control and to significantly increase basal progesterone secretion by 61%, while PGF-2 had no effect. On day-9 CL, PGE-2 was ineffective, but PGF-2 caused a 2·5-fold increase of NOS activity and a marked decrease in progesterone production. Using specific inhibitors, we found that the regulatory actions of PGE-2 in vitro are mediated via the adenyl cyclase/protein kinase A (PKA) second messenger system, while the PGF-2 -induced luteolytic effects on day-9 CL depend upon activation of the phospholipase C/protein kinase C (PKC) system. The different responsiveness of day-4 and day-9 CL to PGE-2 and PGF-2 could depend on receptor availability for these two prostaglandins, even if other cellular mechanisms cannot be excluded. The present study supports a functional role for NOS in regulating the steroidogenic capacity of rabbit CL, and reveals a novel interaction between a stimulatory G-protein-coupled receptor and PKC/PKA-mediated signal transduction modulating NOS activity.

The aim of this study was to better understand the role of the endothelin-1 (ET-1) system in the process of controlling the corpora lutea (CL) life span in rabbits. ET-1 (10 microg iv) administration at d 9 and 12 of pseudopregnancy induced a functional luteolysis within 24 h of injection, but it was ineffective at both d 4 and 6. Pretreatments with Bosentan, a dual ET(A)/ET(B) receptor antagonist, or cyclooxygenase (COX) inhibitor blocked the luteolytic action of ET-1 but not that induced by prostaglandin F2alpha (PGF2alpha). In CL cultured in vitro, ET-1 increased (P

Total activity of nitric oxide synthase (NOS) and the gene expression of both endothelial NOS (eNOS) and inducible NOS (iNOS) isoforms in corpora lutea of pseudopregnant rabbits were examined during prostaglandin F 2␣ (PGF 2␣ )-induced luteolysis. Corpora lutea were collected at 0, 6, 12, 24 and 48 h after an injection of PGF 2␣ at day 9 of pseudopregnancy. At 12 h after PGF 2␣ administration, luteal mRNA encoding eNOS decreased (P 0.05) by 40% and remained low throughout the subsequent 36 h, whereas eNOS protein increased (P 0.05) two-to threefold. By contrast, expression of mRNA encoding iNOS was poor and remained fairly constant, but transcription increased eightfold (P 0.01) within 6 h after PGF 2␣ treatment and then decreased to values similar to those of controls. Total NOS activity increased twofold (P 0.01) at 6 h after treatment and remained high thereafter, whereas progesterone concentrations in explanted corpora lutea decreased (P 0.01) from 302.4 ± 42.3 pg mg −1 at day 9 to 58.6 ± 8.3 at 48 h later, and peripheral plasma concentrations of progesterone declined too. Long-term administration of N -nitro-L-arginine methyl ester (0.6 g l −1 per os) from day 2 of pseudopregnancy onward partially blocked the luteolytic action of PGF 2␣ administered at day 9 of pseudopregnancy. In nitric oxide (NO)-deficient rabbits, progesterone concentrations remained higher (P 0.01) than in controls at 24-48 h after PGF 2␣ administration (4.5 to 3.2 ng ml −1 , respectively). These data are the first to characterize NOS activity. The time course of expression of eNOS and iNOS in rabbit corpora lutea during PGF 2␣ -induced luteolysis gives additional support to a physiological role of NO in the regulation of regression of corpora lutea in rabbits.

There is emerging evidence that microRNAs (miRNAs) dysregulation is involved in the genesis and the progression of Prostate Cancer (PCa), thus potentially increasing their use in urological clinical practice. This is the first pilot study which utilizes Illumina Deep Sequencing to examine the entire miRNAs spectrum existent in urine exfoliated prostate cells (UEPCs) of PCa patients. A total of 11 male patients with histological diagnosis of PCa were enrolled in the present study. First-catch urine (30 mL) was collected following a prostate massage. Total RNA was extracted from urine and sequenced using an HiSeq2500 System (Illumina). QPCR assay was used to validate the highest NGS results in PCA patients and in age-matched, caucasian men. Remarkably, PCA let-7 family was down-regulated (P < 0.01), compared to the controls. The results of our study support the notion of a relatively high diagnostic value of miRNA family for PCa detection, especially in the let-7 family. The present research confirmed the potential use of miRNAs as non-invasive biomarkers in the diagnosis of PCa, potentially reducing the invasiveness of actual clinical strategy.

We aimed at assessing the stability of candidate reference genes in urine sediments of men subjected to digital rectal examination for suspected prostate cancer (PCa). Two microRNAs (miR-191 and miR-25) and 1 small nucleolar RNA (SNORD48) were assayed in 35 post-DRE urine sediments of men with PCa and in 26 subjects with histologically confirmed benign prostatic hyperplasia (BPH). The stability of candidate reference genes was assessed through BestKeeper algorithm and equivalence test. miR-200b and miR-452 were used to test for the effect of normalization on target genes. Our results proved miR-191 to be the most stable gene, showing the lowest degree of variation and the highest stability value. miR-25 and SNORD48 values fell beyond the cutoff of acceptability. In conclusion, we recommend the use of miR-191 for normalization purposes in post-DRE urine sediments.