Treatment by testicular sperm extraction and intracytoplasmic sperm injection of 65 azoospermic patients with non-mosaic Klinefelter syndrome with birth of 17 healthy children.

The aim of this work was to present the clinical and embryological outcomes of 65 azoospermic patients with non-mosaic Klinefelter syndrome (KS), treated by testicular sperm extraction (TESE), followed by intracytoplasmic sperm injection (ICSI), either with fresh or cryopreserved testicular spermatozoa. In total, spermatozoa were recovered in 25/65 (38.5%) of the cases. Of the 48 patients who choose to perform TESE followed by ICSI using fresh testicular spermatozoa (treatment TESE), spermatozoa was recovered in 19 patients (40%), with birth of 12 newborn. Of the 17 patients who choose to perform TESE followed by testicular sperm cryopreservation, spermatozoa were recovered in six patients (35%), with birth of one child. Of the patients who performed treatment TESE, nine went for a new cycle using cryopreserved spermatozoa. Of these, five patients had a previous failed treatment cycle (two patients, three newborn) and four with a previous success went for a new cycle (one patient, one newborn). Overall, the embryological and clinical rates were as follows: 52% of fertilization, 41% of blastocyst, 27% of implantation, 39% of live birth delivery and 47% of newborn. Of the 16 clinical pregnancies, 14 had a successful delivery (12 girls and 5 boys). The 17 newborns had a mean gestation time of 37.2 weeks (35.3% pre-term) and a mean newborn weight of 2781.3 g (37.5% low weight). Comparisons between cycles with fresh and frozen-thaw spermatozoa revealed higher fertilization and clinical pregnancy rates with fresh spermatozoa, with no differences regarding implantation or newborn rates. Of the 17 newborns, no abnormal karyotypes (n = 3) or numerical abnormalities in chromosomes 13, 18, 21, X and Y (n = 14) as evaluated by Multiplex Ligation-dependent Probe Amplification were observed. In conclusion, this study presents further data that reassures that men with KS have no increased risk of transmitting their genetic problem to the offspring.

Renal aging in WKY rats: changes in Na+,K+ -ATPase function and oxidative stress.

It has been suggested that alterations in Na(+),K(+)-ATPase mediate the development of several aging-related pathologies, such as hypertension and diabetes. Thus, we evaluated Na(+),K(+)-ATPase function and H(2)O(2) production in the renal cortex and medulla of Wistar Kyoto (WKY) rats at 13, 52 and 91 weeks of age. Creatinine clearance, proteinuria, urinary excretion of Na(+) and K(+) and fractional excretion of Na(+) were also determined. The results show that at 91 weeks old WKY rats had increased creatinine clearance and did not have proteinuria. Despite aging having had no effect on urinary Na(+) excretion, urinary K(+) excretion was increased and fractional Na(+) excretion was decreased with age. In renal proximal tubules and isolated renal cortical cells, 91 week old rats had decreased Na(+),K(+)-ATPase activity when compared to 13 and 52 week old rats. In renal medulla, 91 week old rats had increased Na(+),K(+)-ATPase activity, paralleled by an increase in protein expression of α(1)-subunit of Na(+),K(+)-ATPase. In addition, renal H(2)O(2) production increased with age and at 91 weeks of age renal medulla H(2)O(2) production was significantly higher than renal cortex production. The present work demonstrates that although at 91 weeks of age WKY rats were able to maintain Na(+) homeostasis, aging was accompanied by alterations in renal Na(+),K(+)-ATPase function. The observed increase in oxidative stress may account, in part, for the observed changes. Possibly, altered Na(+),K(+)-ATPase renal function may precede the development of age-related pathologies and loss of renal function.

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity.

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 +/- 3.43 nmol p-nitrophenol.mg protein-1.min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). beta-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 ± 3.43 nmol p-nitrophenol·mg protein-1·min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84 percent, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11 percent, respectively). â-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36 percent, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18 percent) and inhibited (13 percent) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

Expression and function of LAT1, a neutral amino acid exchanger, in renal porcine epithelial cell line LLC-PK.

AIM: The present study examined the expression of LAT1 and the functional characteristics of the inward and outward [14C] l-leucine transporter in the renal porcine epithelial cell line LLC-PK1. METHODS: LLC-PK1 cells were cultured in polycarbonate filters and accumulation and transepithelial flux of the substrate monitored with [14C] l-leucine. LAT1 transcripts were examined by RT-PCR. LAT1 protein was detected by immunoblotting. RESULTS: The accumulation of [14C] l-leucine in the cell and the [14C] l-leucine transepithelial flux were four- and twofold, respectively, when the substrate was added from the basal cell side, suggesting that the basolateral membrane is endowed with a high density of transport units, when compared with the apical membrane. Increases in the transepithelial flux of [14C] l-leucine by unlabelled l-leucine were also more pronounced when unlabelled l-leucine was added from the basolateral membrane. In the absence of Na+, unlabelled l-leucine increased the basal and apical fractional outflow of [14C] l-leucine, this being similar at pH 7.4 and pH 6.2. RT-PCR and immunoblotting detected LAT1 transcript and protein, respectively. CONCLUSION: LLC-PK1 cells are endowed with the LAT1 transcript and protein and transport l-leucine through the Na+-independent and pH-insensitive LAT1 transporter. The density of transporter units in LLC-PK1 cells may be higher at the basolateral membranes, although be also present in the apical membranes.

Expression of LAT1 and LAT2 amino acid transporters in human and rat intestinal epithelial cells.

The present study evaluated the presence of LAT1 and LAT2 amino acid transporters in human Caco-2 cells and rat IEC-6 cells along the mucosa of the rat digestive tract. The LAT1 cDNA was amplified by PCR using two sets of primers (one specific for rat LAT1 and another simultaneously specific for human, rat and mice). The LAT2 cDNA was amplified by PCR using one set of primers simultaneously specific for human, rat and mice LAT2. The presence of LAT1 and LAT2 protein was examined by means of immunoblotting using an antibody raised against the rat LAT1 and mouse LAT2. Caco-2 and IEC-6 cells, as well as the rat intestinal mucosa, are endowed with both LAT1 and LAT2 transporter transcripts and protein. LAT1 protein is most abundant in IEC-6 cells, which is in agreement with functional data previously reported. The findings in the rat intestinal mucosa indicate that LAT1 protein is most abundant in the colon and its abundance markedly decreases at the level of jejunum and ileum, which contrast with relative homogeneous presence of LAT2 across the digestive tract. In conclusion, Caco-2 and IEC-6 cells, as well as the rat intestinal mucosa, are endowed with both LAT1 and LAT2 amino acid transporter transcripts and protein.