A new media without animal component for sperm cryopreservation: motility and various attributes affecting paternal contribution of sperm.

PURPOSE: Our aim was the development of a safe sperm cryopreservation New Media (NM), composed of consistent and reproducible components devoid of any animal origin, and evaluation of NM in terms of its effect on sperm structure and function as compared to regularly used yolk media (TYM) (Irvine Scientific). METHODS: We evaluated patient semen samples and cryopreserved them in duplicates in either NM or TYM. The samples were cryopreserved for either a short term of 1 week or long term of 1 month prior to thawing. The parameters investigated include sperm motility via computer-assisted semen analysis (CASA), sperm concentration, and sperm biomarkers that promote paternal contribution of spermatozoa to fertilization including hyaluronic acid binding, chromatin maturity, apoptotic markers, cytoplasmic retention, and sperm DNA integrity. RESULTS: As compared to TYM, NM was equally capable of sperm cryopreservation with both short-term and long-term storage in media, and after freeze-thaw and gradient processing of sperm. HA binding of sperm was comparable post thaw in both NM and yolk media. There are also no differences observed between the samples cryopreserved in NM or TYM in terms of their aniline blue staining, CK immunocytochemistry, caspase 3 immunostaining, or DNA nick translation. CONCLUSIONS: NM has the advantage of being xeno-free, yet in preservation of sperm motility and other sperm attributes, the NM is as effective as the TYM.

Effects of abamectin exposure on male fertility in rats: potential role of oxidative stress-mediated poly(ADP-ribose) polymerase (PARP) activation.

Despite the known adverse effects of abamectin pesticide, little is known about its action on male fertility. To explore the effects of exposure to abamectin on male fertility and its mechanism, low (1mg/kg/day) and high dose (4 mg/kg/day) abamectin were applied to male rats by oral gavage for 1week and for 6weeks. Weight of testes, serum reproductive hormone levels, sperm dynamics and histopathology of testes were used to evaluate the reproductive efficiency of abamectin-exposed rats. Abamectin level was determined at high concentrations in plasma and testicular tissues of male rats exposed to this pesticide. The testes weights of animals and serum testosterone concentrations did not show any significant changes after abamectin exposure. Abamectin administration was associated with decreased sperm count and motility and increased seminiferous tubule damage. In addition, significant elevations in the 4-hydroxy-2-nonenal (4-HNE)-modified proteins and poly(ADP-ribose) (PAR) expression, as markers for oxidative stress and poly(ADP-ribose) polymerase (PARP) activation, were observed in testes of rats exposed to abamectin. These results showed that abamectin exposure induces testicular damage and affects sperm dynamics. Oxidative stress-mediated PARP activation might be one of the possible mechanism(s) underlying testicular damage induced by abamectin.

Histopathologic evaluation of pulpal tissue response to various adhesive cleanup techniques.

INTRODUCTION: The aim of this prospective in-vivo study was to investigate the possible effects of temperature changes from various adhesive cleanup procedures on pulpal tissue. METHODS: The materials, consisting of 40 sound maxillary and mandibular premolars to be extracted during orthodontic treatment, were randomly assigned to 4 groups, with 1 group as the control. The teeth in the 3 study groups were etched; brackets were bonded and then debonded. The remaining adhesive was removed with a tungsten carbide bur by using a high-speed hand piece. The teeth in the control group were not etched and bonded. In group 1, the residual adhesive was removed with water cooling, and the teeth were extracted 24 hours later. In group 2, the residual adhesive was removed without water cooling, and the teeth were extracted 24 hours later. In group 3, the residual adhesive was removed without water cooling, and the teeth were extracted 20 days later. The teeth were prepared for histologic examination, and the number of vessels, vessel areas and perimeters, extravasation of red blood cells, vascular congestion, and inflammatory cell infiltration were evaluated to determine pulpal tissue changes. RESULTS: According to the findings from histologic and immunohistochemical evaluations, the coronal pulps of the teeth in groups 1 and 3 were almost similar to the control teeth, but some distinct pathologic changes were observed in group 2. CONCLUSIONS: Adhesive removal without water cooling caused some vascular and pulpal tissue alterations, but these were tolerated by the pulpal tissues, so the changes were reversible.

Role of TNF-related apoptosis-inducing ligand (TRAIL) in the pathogenesis of varicocele-induced testicular dysfunction.

The higher frequency of varicocele in men with infertility has drawn attention and resulted in increased research at the molecular level towards treatments. The aim of this study was to investigate the role of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors in varicocele-induced testicular dysfunction in an experimental rat model. The rats were divided into three groups: control, sham and varicocele. Varicoceles in rats were induced by partial ligation of the left renal vein and left testes. The rats were analyzed 13 weeks after surgery. The degree of DNA fragmentation within cells in the testis was determined using terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay. Tubule degeneration was evaluated using the Johnsen score. The expression of TRAIL and its receptors was detected by immunohistochemical and Western blotting techniques. The apoptotic index, Johnsen score and the expression of TRAIL and TRAIL receptors were examined. The data are presented as the mean±s.d. and were analyzed using computer software. The Kruskal-Wallis and Dunn's multiple comparison tests were used in the statistical analyses. The germ cell apoptotic index was increased in rats with varicoceles when compared with the sham and control groups (P=0.0031). The Johnsen score was significantly decreased in the varicocele group when compared with the sham and control groups (P<0.0001). Immunohistochemical and Western blotting analyses showed that after varicocele induction, the expression of TRAIL-R1 and TRAIL-R4 in germ cells was increased and the expression of TRAIL-R2 was decreased. There are no significant differences among the groups in terms of TRAIL and TRAIL-R3 receptor expression. The results of this study indicate that TRAIL and its receptors may have a potential role in the pathogenesis of varicocele-induced testicular dysfunction.

Potential role of poly(ADP-ribose) polymerase activation in the pathogenesis of experimental left varicocele.

The aim of this study was to evaluate whether the poly(adenosine diphosphate[ADP]-ribose) polymerase (PARP) pathway is activated by experimental left varicocele. Rats underwent partial ligation of the left renal vein to induce experimental varicocele, and left testes were analyzed 13 weeks after surgery. Tubule degeneration was evaluated by Johnsen score. Expression of 4-hydroxy-2-nonenal (4-HNE)-modified proteins, PARP-1, and poly(-ADP-ribose) (PAR) was detected by immunohistochemistry and Western blot. The degree of apoptosis within testes was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling. Light microscopy revealed testicular damage comprising various degrees of seminiferous tubule degeneration. Germ cell apoptotic index and 4-HNE, PAR, and PARP-1 expression in germ cells increased after varicocele induction. Increased oxidative stress and PARP overactivation in testes might be important with regard to impaired testicular function associated with varicocele.

Role of the poly(ADP-ribose)polymerase activity in vancomycin-induced renal injury.

The aim of the present study was to investigate the role of poly(ADP-ribose)polymerase (PARP) activity in vancomycin (VCM)-induced renal injury and to determine whether 1,5-isoquinelinediol (ISO), a PARP inhibitor agent, could be offered as an alternative therapy in VCM-induced renal impairment. Rats were divided into four groups as follows: (i) control (Group 1); (ii) VCM-treated (Group 2); (iii) VCM plus ISO-treated (Group 3); and (iv) ISO-treated (Group 4). VCM (200mg/kg, i.p., twice daily) was administered to Groups 2 and 3 for 7 days. ISO (3mg/kg/day, i.p.) treatment was started 24h before the first administration of VCM and continued for 8 days. After the 14th VCM injection, the animals were placed in metabolic cages to collect urine samples. All the rats were sacrificed by decapitation, blood samples were taken in tubes and kidneys were excised immediately. Blood urea nitrogen (BUN) and plasma creatinine, and urinary N-acetyl-beta-d-glucosaminidase (NAG, a marker of renal tubular injury) were used as markers of VCM-induced renal injury in rats. Light microscopy was used to evaluate semi-quantitative analysis of the kidney sections. Poly(ADP-ribose) (PAR, the product of activated PARP) and PARP-1 expressions in renal tissues were demonstrated by immunohistochemistry and Western blot. VCM administration increased BUN levels from 8.07+/-0.75 mg/dL to 53.87+/-10.11 mg/dL. The plasma creatinine levels were 0.8+/-0.04 mg/dL and 3.38+/-0.51 mg/dL for the control and VCM-treated groups, respectively. Also, urinary excretion of NAG was increased after VCM injection. Besides, there was a significant dilatation of the renal tubules, eosinophilic casts within some tubules, desquamation and vacuolization of renal tubule epithelium, and interstitial tissue inflammation in VCM-treated rats. In VCM-treated rats, both PAR and PARP-1 expressions were increased in renal tubular cells. ISO treatment attenuated VCM-induced renal injury, as indicated by BUN and plasma creatinine levels, urinary NAG excretion, and renal histology. PARP inhibitor treatment also decreased PAR and PARP-1 protein expressions similar to that of controls. Herewith, the overactivation of the PARP pathway may have a role in VCM-induced renal impairment and pharmacological inhibition of this pathway might be an effective intervention to prevent VCM-induced acute renal injury.

Presence of the brain proteins cerebral cavernous malformation-2 and cerebral cavernous malformation-3 in rat testes and their potential role in experimental varicocele.

OBJECTIVE: To assess the distribution of cerebral cavernous malformation-2 (CCM2) and CCM3 proteins in a normal and experimentally induced varicocele model in rat testes. DESIGN: Comparative and controlled study. SETTING: University animal care and operation unit. ANIMAL(S): Wistar male rats for experimental and control groups. INTERVENTION(S): The control group underwent a sham operation. Rats in the experimental groups underwent partial ligation of the renal vein to induce experimental varicocele, and left testicular tissues were analyzed. MAIN OUTCOME MEASURE(S): Tissues were fixed and processed for paraffin-embedded testicular tissues. Levels of CCMs were assessed by immunohistochemistry and Western blot analysis. RESULT(S): In control and sham-operated testes, CCM2 expression was detected in acrosomes of round spermatids at stages 6-8 and in the heads of elongating spermatids at stages 1-5 and 9-14. CCM3 expression was weakly localized in pachytene spermatocytes at stages 8-12 and strongly immunolocalized in the cytoplasm of round spermatids at stages 1-8 and in elongating spermatid cytoplasm at stages 9-12. Varicocele induction clearly demonstrated histopathological changes due to germ cell degeneration. CCM2 and CCM3 expression increased significantly in varicocele-induced rat testes. CONCLUSION(S): The expression patterns of two brain proteins were clearly identified in rat testes, suggesting a role during normal and pathological spermatogenesis. This article represents the first demonstration that these proteins have a role outside the central nervous system.

Aldosterone-induced endothelial dysfunction of rat aorta: role of poly(ADP-ribose) activation.

INTRODUCTION: The aim of this study was to investigate whether activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) contributes to the development of aldosterone-induced endothelial dysfunction and treatment with the potent PARP inhibitor 1,5-isoquinolinediol (3 mg/kg/day, i.p.) could prevent endothelial dysfunction caused by aldosterone. METHODS: Infusion of subpressor doses of aldosterone with subcutaneously implanted mini-osmotic pumps (0.05 mg/kg/day) to rats for 21 days induced the development of endothelial dysfunction. In order to evaluate endothelial function, isometric tension studies were performed in response to acetylcholine and sodium nitroprusside.Additionally, PAR (the end product of activated PARP) and PARP-1 expressions in the endothelium of thoracic aortas were evaluated by immunohistochemistry. RESULTS: There was a significant loss of endothelium-dependent vasodilatation in response to acetylcholine in aldosterone-infused rats. In animals treated with 1,5-isoquinolinediol, the effect of aldosterone on vascular responsiveness was less than the untreated groups. Immunohistochemical studies demonstrated that aldosterone administration increased PAR and PARP-1 expressions in the endothelium of thoracic aortas, whereas PARP inhibition decreased their expressions to control levels. CONCLUSION: Our results indicate that PARP activation in the vascular system may be a contributory factor to the impaired endothelial function associated with aldosterone administration.