Accumulation of protoporphyrin-IX in rat Leydig cells following induction by 5-aminolevulinic acid and tramadol.

OBJECTIVES: This study aimed to monitor the accumulation of endogenous protoporphyrin-IX (PpIX) in rat Leydig cells (R(2)C) under the effect of 5-aminolevulinic acid (ALA) and various concentrations of tramadol, an analgesic drug. BACKGROUND DATA: Pain during photodynamic treatment with ALA is one of the adverse effects of this new treatment to eradicate tumor cells. ALA is utilized in photodynamic diagnosis and therapy (PDT) as a compound capable of augmenting the intracellular pool of PpIX, which exhibits properties of a photosensitizer. METHODS: Cellular content of PpIX was determined following incubation of the cells for 1 and 2 h in culture medium that contained ALA and different concentrations of tramadol. The amount of PpIX was determined using fluorescent technique under a confocal microscope (laser wavelength 458 nm and filter LP 585 nm), and evaluated using CytFlu 1.2 software. RESULTS: After 1 h of incubation, no significant alterations were noted in the cellular PpIX concentration. However, 2 h of incubation resulted in a significant increase (p < 0.05) in PpIX fluorescence inside the cells, when the medium contained ALA and tramadol in concentrations ranging from 1-2 mg/1 mL. CONCLUSIONS: The results suggested that in R(2)C cells, exogenous ALA and tramadol induced protoporphyrin accumulation. This information is useful for two reasons. First, it may help to diminish pain after ALA-PDT treatment; and second, it allows the use of lower concentrations of ALA during therapy.

Human spermatozoa ultrastructure assessment in the infertility treatment by assisted reproduction technique.

The aim of this study was to prospectively investigate the spermatozoa ultrastructure in relation to the results of in vitro fertilization-embryo transer (IVF-ET). Forty-nine consecutive couples admitted for IVF-ET were prospectively evaluated for electron microscopic spermatozoa morphology and the outcome of IVF-ET. Thirty-four couples revealed successful fertilization, defined as presence of two pronuclei 14-16 hours after spermatozoa administration, while the remaining 15 formed the failure group. Spermatozoa fixed with 2.5% glutaraldehyd and embedded in Spurr's resin were analyzed with JAM 100 S transmission electron microscope (TEM) for the following ultrastructure abnormalities: head deformity, cytoplasmic residues, chromatin condensation failures, acrosomal alterations, neck defects, mid-piece defects, principal piece and end-piece defects and immature forms. Successful IVF-ET couples revealed a significantly higher percentage of normal spermatozoa utrastructure (32.0 +/- 13.1% versus 17.1 +/- 13.4%, p < 0.001). Failed IVF-ET couples represented a significantly higher percentage of chromatin condensation failures (9.8 +/- 5.1% versus 5.7 +/- 5.3%, p < 0.05) and tail defects (16.7 +/- 11.5% versus 7.2 +/- 7.2%, p < 0.001). A positive correlation between normal ultrastructure spermatozoa percentage and fertilized oocytes percentage was found (r = 0.35, p < 0.05). Our data suggest that spermatozoa TEM findings correlate with IVF-ET results. Ultrastructural estimation of spermatozoa can improve the diagnosis of male fertility and may explain some reasons of failure in assisted reproduction methods. We consider systematic TEM spermatozoa examination in cases with failed IVF-ET prior to intracytoplasmic sperm injection (ICSI).

Effects of galanin on proliferation and apoptosis of immature rat thymocytes.

Evidence indicates that some regulatory peptides (endothelins, cholecystokinin and VIP) are involved in the control of thymus growth, and we have investigated whether galanin may be included in this group of peptides. In fact, galanin, a 29-amino acid peptide acting through three subtypes of G protein-coupled receptors (GalR1, GalR2 and GalR3), seems to play a role in the control of the immune system. Reverse transcription (RT)-polymerase chain reaction (PCR) allowed the detection of galanin, GalR1 and GalR3 mRNAs in the thymus cortex of immature (20-day-old) rats, while GalR2 expression was very weak or absent. Immature rats were given three subcutaneous injections (28, 16 and 4 h before sacrifice) of 2 nmol/100 g galanin and or the galanin-receptor antagonist (galanin-A) [D-Thr(6),D-Trp(8,9),15-ol]-galanin(1-15), and 0.1 mg/100 g vincristine 3 h before sacrifice. Thymuses were processed for light microscopy and the percentage of metaphase-arrested cells (mitotic index) was evaluated. Galanin-A increased the thymus mitotic index, while galanin was ineffective, thereby suggesting that endogenous galanin exerts a maximal tonic inhibitory effect on the proliferative activity of thymocytes in immature rats. Immature rat thymocytes were cultured in vitro for 12 h in the presence of 10(-6) M galanin and/or galanin-A. Hoechst 33342 and propidium iodide were added to the cultures, and the percentage of apoptotic and necrotic cells was determined under fluorescence microscope. Galanin increased apoptotic index, and the effect was prevented by galanin-A. Neither galanin nor galanin-A altered necrotic index. Collectively, our findings indicate that galanin, probably acting through GalR1 and GalR3, exerts antiproliferative and proapoptotic effects on immature rat thymocytes, which makes it likely that this peptide plays a role in the autocrine/paracrine functional regulation of immune system in the rat.