Comparison of the effects of Ham'sF10 and αMEM in combination with FBS or BSA in vitrification/warming solution on quality and viability of sheep ovarian follicles.

The aim of this study was to evaluate the effects of the two types of media, namely minimum essential medium (αMEM) and Ham'sF10, supplemented with foetal bovine serum (FBS) or bovine serum albumin (BSA) in vitrification/warming solution on the quality and viability of sheep ovarian follicles. Vitrification method was applied for cryopreservation of sheep ovarian cortex using Ham'sF10 and αMEM supplemented with either BSA or FBS. There were five groups: Fresh, Ham'sF10+ BSA, Ham'sF10+ FBS, αMEM + BSA and αMEM + FBS. Samples were cultured for two weeks after warming. Viability and morphology of follicles and DNA fragmentation in follicles and in tissue stroma cells were analysed before vitrification/warming and following one and two weeks of culture. The Ham'sF10+ FBS and Ham'sF10+ BSA groups showed a significant decrease in follicular viability after one week of culture (p < .05 vs. Fresh). Following two weeks of culture, all groups revealed a considerable fall in the number of viable follicles (p < .05 vs. Fresh). There was an increase in DNA fragmentation of connective tissue cells but not in the follicles (p < .05). Our results showed the better application of αMEM supplemented with BSA as a vitrification solution in improvement of cryopreservation effects and maintenance of follicular survival.

Protective features of resveratrol on human spermatozoa cryopreservation may be mediated through 5' AMP-activated protein kinase activation.

Biochemical and physical modifications during the freeze-thaw process adversely influence the restoration of energy-dependent sperm functions required for fertilization. Resveratrol, a phytoalexin, has been introduced to activate 5' AMP-activated protein kinase which is a cell energy sensor and a cell metabolism regulator. The cryoprotection of resveratrol on sperm cryoinjury via activation of AMP-activated protein kinase also remains to be elucidated. Our aim, thus, was to investigate: (i) the presence and intracellular localization of AMP-activated protein kinase protein; (ii) whether resveratrol may exert a protective effect on certain functional properties of fresh and post-thaw human spermatozoa through modulation of AMP-activated protein kinase. Spermatozoa from normozoospermic men were incubated with or without different concentrations of Compound C as an AMP-activated protein kinase inhibitor or resveratrol as an AMP-activated protein kinase activator for different lengths of time and were then cryopreserved. AMP-activated protein kinase is expressed essentially in the entire flagellum and the post-equatorial region. Viability of fresh spermatozoa was not significantly affected by the presence of Compound C or resveratrol. However, although Compound C caused a potent inhibition of spermatozoa motility parameters, resveratrol did not induce negative effect, except a significant reduction in motility at 25 µm for 1 h. Furthermore, resveratrol significantly increased AMP-activated protein kinase phosphorylation and mitochondrial membrane potential and decreased reactive oxygen species and apoptosis-like changes in frozen-thawed spermatozoa. Nevertheless, it was not able to compensate decreased sperm viability and motility parameters following cryopreservation. In contrast, Compound C showed opposite effects to resveratrol on AMP-activated protein kinase phosphorylation, reactive oxygen species, apoptosis-like changes, mitochondrial membrane potential, and motility parameters. These findings, although preliminary, suggest that resveratrol-induced improvement of cryopreserved sperm functions may be mediated through activation of AMP-activated protein kinase, indicating the importance of AMP-activated protein kinase activity for human spermatozoa functions. Further investigations are required to elucidate the mechanism by which resveratrol ameliorates oxidative stress-mediated damages in an AMP-activated protein kinase-dependent mechanism.

Effect of brain-derived neurotrophic factor on sperm function, oxidative stress and membrane integrity in human.

Oxidative stress has negative impacts on the clinical outcomes of assisted reproduction techniques. The brain-derived neurotrophic factor (BDNF) promotes the viability of nerve cells and is known to decrease oxidative stress and apoptosis in different cells. The aim of this study was to evaluate the effect of BDNF treatment on human sperm functions that are known to be essential for fertilisation. Our findings showed that treatment of human spermatozoa with 0.133 nM BDNF significantly increased the percentages of both total (P = 0.001) and progressive (P < 0.01) motile sperm cells compared to those observed in the nontreated (control) group. We also showed that the mean fluorescence intensity of DCFH-DA, as an indicator of intracellular reactive oxygen species, was significantly lower (P < 0.05) in spermatozoa treated with BDNF compared to the control group. Treatment of spermatozoa with BDNF significantly decreased the percentages of both dead (P = 0.001) and apoptotic-like sperm cells (P < 0.05) compared to the control group. On the other hand, BDNF treatment significantly increased the percentage of viable sperm cells compared to the control (P = 0.001). In conclusion, BDNF has protective effects against oxidative stress in spermatozoa and could improve sperm functions that are essential for sperm-egg fusion and subsequent fertilisation.

Efficacy of Crocus sativus L. on reduction of cadmium-induced toxicity on spermatogenesis in adult rats.

Cadmium is a toxic heavy metal element, which probably cause infertility by impairment in spermatogenesis. The present work aimed (i) to study the toxic effect of cadmium on spermatogenesis in rat, as well as (ii) the protective effect of Crocus sativus L. on cadmium-intoxicated rats. Cadmium chloride was administered intraperitoneally during 16 days at intervals of 48 h between subsequent treatments. Crocus sativus L. was pre-treated in both of control and cadmium-injected rats. Animals were sacrificed on day 17 after the first treatment. The left cauda epididymis was removed and immediately immersed into Hank's balanced salt solution for the evaluation of sperm count and viability, and left testis was fixed in 10% formalin for histological evaluation. Following contamination with cadmium, a decrease was observed in the number and viability of cauda epididymis sperm, which were increased by Crocus sativus L. pre-treatment (P < 0.05). In addition, cadmium decreased both cell proliferation and Johnsen Scores in the seminiferous tubules, which were reversed by Crocus sativus pre-treatment (P < 0.05). Furthermore, cadmium-induced decrease in the amount of free serum testosterone as well as an increase in lipid peroxidation activity in the testicular tissue was reversed by Crocus sativus L. (P < 0.05). These findings may support the concept that Crocus sativus L. can improve the cadmium toxicity on spermatogenesis.

Planning screw insertion trajectory in lumbar spinal fusion using pre-operative CT images.

Spinal fusion permanently connects two or more vertebrae in spine to improve stability, correct a deformity or reduce pain by immobilizing the vertebrae through pedicle screw fixation. Pedicle screws should be inserted very carefully to prevent possible irrecoverable damages to the spinal cord. Surgeons use CT/fluoroscopic images to find how to insert the screws safely. However, there is still human error, as determining precise trajectory in 3D space is difficult because of asymmetric structure of pedicle. In this study we attempt to propose a shape based method to help the surgeons to find the more accurate and safe path for screw insertion that minimizes the risk or invasiveness of the surgery using pre-operative CT images. We extracted two features for insertion paths from CT images, named "safety margin" and "pedicular screw fixation strength". By using weighted k-means different paths were clustered and compared with each other. Results of comparison between those paths obtained from surgeon's pre-operative planning, intra operative and the proposed method proves a great improvement on the rate of success in reaching a suitable insertion trajectory by using our method. It is observed that the risk of damage in intra operative stage can be potentially high and it can be reduced considerably by using the proposed planning approach.

Effect of aminoguanidine in sperm DNA fragmentation in varicocelized rats: role of nitric oxide.

BACKGROUND AND PURPOSE: The sperm of infertile men with varicocele exhibit markedly high DNA damage that appears to be related to high oxidative stress (OS). Aminoguanidine (AG) is a specific inhibitor of the nitric oxide synthase (NOS) isoforms iNOS and an antioxidant, the effects of which decrease NO and peroxynitrite production. The aim of this study was to determine the effects of AG on sperm chromatin in varicocelized rats. METHODS: Thirty male Wistar rats were divided into 5 groups: control, sham, varicocele, and AG and placebo-treated groups. At 10 weeks after varicocele induction, sperm chromatin was evaluated in all groups, except in the treated groups. The treated groups received intraperitoneal injections of 50 mg/kg AG or placebo daily for 10 weeks and then were killed for chromatin assessment. Sperm chromatin was evaluated by aniline blue, acridine orange, toluidine blue, and chromomycin A(3) staining. RESULTS: The results of the 4 above tests were significantly increased between varicocele and control (and sham) groups (P < .05). CONCLUSION: The findings of this study suggest that AG improves sperm DNA fragmentation that is associated with infertility in varicocelized rats, and treatment with AG can reduce the damage to sperm DNA.

Aminoguanidine improves epididymal sperm parameters in varicocelized rats.

INTRODUCTION: Increased levels of nitric oxide (NO) in the spermatic veins of men affected by varicocele have already been reported. But there is no study to discriminate the subtype of catalytic enzyme for synthesis of NO. In this study, aminoguanidine (AG), an inducible NO synthase inhibitor, has been used to investigate its effect on sperm parameters. METHODS: Twenty-four male Wistar rats were divided into four groups. In groups A and B, left experimental varicocele was induced by a 20-gauge needle. Group C (sham) underwent a similar procedure to groups A and B, but the spermatic vein was left intact, and group D served as control group. The animals in group A were killed 10 weeks later and their sperm count, motility, morphology and vitality were evaluated. Group B received 50 mg/kg AG with i.p. injection daily for 10 weeks. RESULTS: Sperm count, motility, morphology and vitality were significantly decreased in group A in comparison to control group (p ≤ 0.05). In group B, sperm parameters improved in comparison to group A (p ≤ 0.01). Group C did not show any significant alterations in sperm parameters compared with control group. CONCLUSION: These findings may support the concept that AG can improve the sperm count, motility, morphology and vitality in infertile rats with varicocele.

Effects of aminoguanidine on infertile varicocelized rats: A functional and morphological study.

UNLABELLED: BACKGROUNDS AND THE PURPOSE OF THE STUDY: Inducible NO synthase activity has been frequently reported in varicose veins. Aminoguanidine is known to inhibit iNOS. The aim of this study was to examine the effects of aminoguanidine on varicocelized rats. METHODS: Male Wistar rats were divided into groups A, B, C, D, E, and F (control group). Groups A, B, C, and D rats underwent left varicocele induction with a 20-gauge needle. Group E (sham) rats underwent a similar procedure, but the renal vein was left intact. Ten weeks after varicocele induction, sperm parameters were evaluated in groups D, E, and F. Groups A and B received 50 mg/kg aminoguanidine or placebo, respectively, daily for 10 weeks. After 10 and 20 weeks of varicocele induction, the fertility outcomes of the experimental groups were evaluated. RESULTS: The values of the sperm parameters did not differ significantly between groups B and D, but were significant when compared with groups F and E (P≤0.05). The values of the sperm parameters of groups F and E showed no significant changes (P≤0.05). The changes between group A and groups B and D were significant (P≤0.05). Ten weeks after varicocele induction, rats of groups A, B, and C were still fertile. After 20 weeks, only half of the rats in group A were fertile. CONCLUSIONS: Aminoguanidine improved the sperm parameters and mating outcomes in vari-cocelized rats.

Placental and membrane aquaporin water channels: correlation with amniotic fluid volume and composition.

OBJECTIVES: To assess the role of aquaporins (AQPs) in the regulation of amniotic fluid (AF) volume, we determined AF volume and composition and placental and fetal membrane AQP expression throughout the second half of murine gestation. METHODS: Pregnant CD1 mice were sacrificed at e10-19 and AF volume and composition determined. Placenta and fetal membranes were screened for AQP gene expression. AQP gene expression was quantified by real-time RT PCR and protein location determined by immunohistochemistry. Changes in AF volume were correlated with AQP expression. RESULTS: Both membranes and placenta demonstrated expression of AQP1, -3, -8 and -9. Advancing gestation was associated with increased AF volume from e10 to e16, with a marked decrease in AF volume from e16 to e19. By immunohistochemistry, AQP1 was localized to placental vessels and AQP3 to trophoblast. AF volume was negatively correlated with fetal membrane AQP1 and placental AQP1 and AQP9 expression, and positively correlated with placental AQP3 expression. CONCLUSION: Changes in AQPs with advancing gestation, and their correlation with AF volume, suggest a role in mediating placental and membrane water flow and ultimately AF volume. AQP1 appears to regulate fetal membrane water flow, and AQP3 is a likely candidate for the regulation of placental water flow.

Ontogenic expression of putative feeding peptides in the rat fetal brain and placenta.

The well-demonstrated "fetal programming" paradigm is based on the observation that environmental changes can reset the developmental path and thus, gene expression during intrauterine development. As appetite-regulatory neural pathways develop in utero, we sought to determine the ontogenic expression of putative orexigenic and anorexigenic feeding-regulatory peptides in the fetal rat brain and placenta during the last third of gestation. Pregnant Sprague-Dawley rats (n = 12) at D14, D16 and D18 were sacrificed and fetal whole brain and placenta removed and examined for mRNA levels of orexigenic (neuropeptide Y (NPY), agouti-related peptide (AgRP)) and anorexigenic (cocaine and amphetamine regulated transcript (CART), pro-opiomelanocortin (POMC)) peptides and leptin receptor (OB-Rb) using real-time reverse transcription polymerase chain reactions (RT-PCR). For adult comparisons, the hypothalamus, cortex and cerebellum from male rats were also examined for feeding peptides. In the fetal brain and placenta, mRNA levels of AgRP decreased 10-fold from D14 to D16 and was undetectable at D18. Appetite inhibitory factors OB-Rb and CART mRNA levels increased from D14 to D18 in the brain and placenta. NPY and POMC expression remained unchanged from D14 to D18. The pattern of expression of feeding regulatory peptides in the fetal brain most closely resembled the expression profile of the adult cerebral cortex. The continued maturation of feeding regulatory mechanisms in late gestation indicates the potential for in utero programming of ingestive behavior.