Expression of TXNRD1, HSPA4L and ATP1B1 Genes Associated with the Freezability of Boar Sperm.

Cryopreservation is associated with increased oxidative stress, which is responsible for sperm damage. We analyzed the effect of cryopreservation on mRNA and protein expression of thioredoxin reductase 1 (TXNRD1), heat shock protein family A (HSP 70) member 4 like (HSPA4L) and sodium/potassium-transporting ATPase subunit beta-1 (ATP1B1) genes in boar sperm with different freezability. Boars were classified as having good and poor semen freezability (GSF and PSF, respectively), according to the assessment of post-thaw sperm motility. Total RNA was isolated from fresh pre-freeze (PF) and frozen-thawed (FT) sperm from five boars of the GSF and PSF groups, respectively. Quantification of TXNRD1, HSPA4L and ATP1B1 gene expression was performed by RT-qPCR analysis. Proteins extracted from sperm were subjected to Western blotting and SDS-PAGE analyses. Poor freezability ejaculates were characterized by significantly higher relative mRNA expression levels of TXNRD1 and HSPA4L in FT sperm compared with the fresh PF sperm. Furthermore, the relative mRNA expression level of ATP1B1 was significantly higher in the fresh PF sperm of the GSF group. Western blotting analysis revealed significantly higher relative expression of TXNRD1 protein in the fresh PF sperm of the GSF group, while HSPA4L protein expression was markedly increased in FT sperm of the PSF group. Electrophoretic and densitometric analyses revealed a higher number of proteins in the fresh PF and FT sperm of the PSF and GSF groups, respectively. The results of this study indicate that ATP1B1 mRNA expression in the fresh PF sperm is a promising cryotolerance marker, while the variations of TXNRD1 and HSPA4L protein expression in the fresh PF or FT sperm provide useful information that may help to elucidate their biological significance in cryo-damage.

Promoter polymorphisms in STK35 and IFT27 genes and their associations with boar sperm freezability.

We have shown that STK35 and IFT27 genes are differentially expressed in spermatozoa from boars with good and poor semen freezability (GSF and PSF, respectively). STK35 is a stress-related gene that is implicated in spermatogenesis, whereas IFT27 is a motility-related gene that is mainly involved in intracellular protein transport. In this study we hypothesized that polymorphic variants in the 5'-flanking regulatory regions of STK35 and IFT27 genes could contribute to differences in semen freezability. We also predicted the interactions of the polymorphic variants with transcription factors on the gene promoter activity, using bioinformatics. The 5'-flanking region sequences of the STK35 and IFT27 were PCR amplified and analyzed by Sanger sequencing method. Protein expression in STK35 and IFT27 was determined in pre-freeze (PF) and frozen-thawed (FT) spermatozoa, using western blotting analysis. Sanger sequencing revealed a single nucleotide polymorphism (SNP) rs327863835 (C > T) in STK35 promoter, while two SNPs (rs337563873, A > T; rs331520020, T > C) were detected in IFT27 promoter. STK35 and IFT27 promoter polymorphisms showed significant allele frequency differences between the GSF and PSF groups. Using bioinformatics approaches, we predicted that SNPs resulted in the generation of additional transcription factor binding sites for NFATC2, ELK1 and GR-ß, which appeared to enhance or repress the promoter activity of STK35 or IFT27 in either freezability group. Wide variations in STK35 and IFT27 protein expression were observed among the boars, however, significantly higher protein expression was detected in IFT27 in FT spermatozoa of the GSF group. We suggest that the upstream variants, detected in STK35 and IFT27 promoters, might regulate the transcriptional activity of the genes by affecting their potential binding of transcription factors. The results indicate that the allelic variants in STK35 and IFT27 could be considered as potential genetic markers for predicting boar sperm freezability.

Gene promoter polymorphisms in boar spermatozoa differing in freezability.

Single nucleotide polymorphisms (SNPs) in the 5'-flanking regulatory regions of genes could affect their expression levels. This is a follow-up study aimed to identify polymorphic variants in the 5'-flanking regulatory regions of genes expressed in boar spermatozoa, and to predict the interactions of such variants with transcription factors (TFs) on the gene promoter activity, using bioinformatics. Five and six boars were classified as having good and poor semen freezability (GSF and PSF, respectively) according to post-thaw (PT) assessment of sperm motility and membrane integrity characteristics. The 5'-flanking region sequences of the 14 genes (FOS, NFATC3, EAF2, FGF-14, BAMBI, RAB33B, CKS2, LARS2, SLC25A16, ACADM, CPT2, CCT3, DTD2 and CCDC85A) were PCR amplified and analyzed by Sanger sequencing method. A total of 32 polymorphic variants were identified in the 5'-flanking regions of the genes, including 4 insertion/deletion (indel) polymorphisms, and 8 unknown (novel) SNPs. Multiple sequence alignment analysis revealed a 26-bp indel variant in the 5'-flanking region of the LARS2 gene, which showed greater protein expression in spermatozoa from boars of the PSF group. It was found that 17 polymorphic variants, observed in the differentially expressed (DE) genes, showed significant allele frequency differences between the GSF and PSF groups. Polymorphic variants in the 5'-flanking regulatory regions of the genes contributed to the decrease or increase in the binding affinity for different testis-specific TFs, such as SMAD1, NF-1, FOXMI, RXRA, STAT4 and C/EBPß. This study provides more insights into the mechanisms responsible for variations in transcriptional activity in promoters of genes expressed in boar spermatozoa. The allelic variants are promising genetic markers for predicting the freezability of boar spermatozoa.

Regulatory Potential of Long Non-Coding RNAs (lncRNAs) in Boar Spermatozoa with Good and Poor Freezability.

Long non-coding RNAs (lncRNAs) are suggested to play an important role in the sperm biological processes. We performed de novo transcriptome assembly to characterize lncRNAs in spermatozoa, and to investigate the role of the potential target genes of the differentially expressed lncRNAs (DElncRNAs) in sperm freezability. We detected approximately 4007 DElncRNAs, which were differentially expressed in spermatozoa from boars classified as having good and poor semen freezability (GSF and PSF, respectively). Most of the DElncRNAs were upregulated in boars of the PSF group and appeared to significantly affect the sperm's response to the cryopreservation conditions. Furthermore, we predicted that the potential target genes were regulated by DElncRNAs in cis or trans. It was found that DElncRNAs of both freezability groups had potential cis- and trans-regulatory effects on different protein-coding genes, such as COX7A2L, TXNDC8 and SOX-7. Gene Ontology (GO) enrichment revealed that the DElncRNA target genes are associated with numerous biological processes, including signal transduction, response to stress, cell death (apoptosis), motility and embryo development. Significant differences in the de novo assembled transcriptome expression profiles of the DElncRNAs between the freezability groups were confirmed by quantitative real-time PCR analysis. This study reveals the potential effects of protein-coding genes of DElncRNAs on sperm functions, which could contribute to further research on their relevance in semen freezability.

Gene Polymorphisms in Boar Spermatozoa and Their Associations with Post-Thaw Semen Quality.

Genetic markers have been used to assess the freezability of semen. With the advancement in molecular genetic techniques, it is possible to assess the relationships between sperm functions and gene polymorphisms. In this study, variant calling analysis of RNA-Seq datasets was used to identify single nucleotide polymorphisms (SNPs) in boar spermatozoa and to explore the associations between SNPs and post-thaw semen quality. Assessment of post-thaw sperm quality characteristics showed that 21 boars were considered as having good semen freezability (GSF), while 19 boars were classified as having poor semen freezability (PSF). Variant calling demonstrated that most of the polymorphisms (67%) detected in boar spermatozoa were at the 3'-untranslated regions (3'-UTRs). Analysis of SNP abundance in various functional gene categories showed that gene ontology (GO) terms were related to response to stress, motility, metabolism, reproduction, and embryo development. Genomic DNA was isolated from sperm samples of 40 boars. Forty SNPs were selected and genotyped, and several SNPs were significantly associated with motility and membrane integrity of frozen-thawed (FT) spermatozoa. Polymorphism in SCLT1 gene was associated with significantly higher motility and plasma membrane integrity of FT spermatozoa from boars of the GSF group compared with those of the PSF group. Likewise, polymorphisms in MAP3K20, MS4A2, and ROBO1 genes were significantly associated with reduced cryo-induced lipid peroxidation and DNA damage of FT spermatozoa from boars of the GSF group. Candidate genes with significant SNP associations, including APPL1, PLBD1, FBXO16, EML5, RAB3C, OXSR1, PRICKLE1, and MAP3K20 genes, represent potential markers for post-thaw semen quality, and they might be relevant for future improvement in the selection procedure of boars for cryopreservation. The findings of this study provide evidence indicating that polymorphisms in genes expressed in spermatozoa could be considered as factors associated with post-thaw semen quality.

[Novelty of vital dyes in ophthalmic surgery]. / Zastosowanie nowych barwników w chirurgii okulistycznej.

The aim of this paper is to present recent developments in the area of novelty of vital dyes in intraocular surgery. The authors present the advantages and disadvantages of several vital dyes currently used in ophthalmic surgery. Vital dyes are used to allow better intraoperative visualization of both the anterior and posterior segments. Indocyanine green and trypan blue are the most frequently used and the most efficacious dyes for staining the important anatomic areas but often are associated with significant side effects. These dyes are used in cataract and vitreo-retinal surgery. Other dyes including rhodamine 6G, E68, bromophenol blue, light green and Chicago blue are still under preclinical assessment.

[Decrease of the postoperative inflammatory reaction during pars plana vitrectomy (PPV) after administration of triamcinolone acetonide]. / Zmniejszenie pooperacyjnego odczynu zapalnego wskutek zastosowania octanu triamcynolonu podczas witrektomii pars plana.

PURPOSE: To prospectively evaluate the effect on postoperative inflammatory reaction and recovery after application of triamcinolone acetonide (TA) during pars plana vitrectomy (PPV), to visualize the vitreous. MATERIAL AND METHODS: Pars plana vitrectomy (PPV) was performed in 45 patients (21 males, 24 females) (29 with retinal detachment, 6 with macular hole, 1 with cystoid macular edema, 3 with diabetic retinopathy, 3 with vitreous haemorrhage, 1 with preretinal membrane, 1 with PVR and 1 with lens luxation). After surgical separation of the posterior vitreous and removal of any visible epiretinal membrane, TA was injected over the posterior pole. For the control group we used 15 patients (10 with retinal detachment, 2 with macular hole, 1 with preretinal membrane, 1 with lens luxation and 1 with vitreous haemorrhage) (10 males, 5 females) treated with PPV but without TA administration. To evaluate the degree of postoperative inflammation and to monitor the dynamics of the blood-aqueous barrier disruption, the laser flare cell meter (Kowa FM-500) was used. RESULTS: Tyndalometric mean values in the control group of eyes recorded 1 day after PPV were 32.41 +/- 6.1 ph/ msec while values in TA-treated group were significantly lower (20.26 +/- 2.4, p < 0.02). 10 days after surgery in TA group results were still significantly lower as compared to the control group (16.4 +/- 2.6 vs 32.5 +/- 9.6, p < 0.005). As observed 6 weeks after PPV, tyndalometric recordings in TA-treated group remained lower as those observed in the control group (16.1 +/- 3.1 vs 32.0 +/- 8.1, p < 0.01). CONCLUSIONS: The eyes which received TA-assisted PPV showed significantly less breakdown of the blood-ocular barrier than those with routine PPV. Intraoperative administration TA facilitates postoperative recovery after surgery lowering the inflammatory reaction.

[Assessment of the macula function by static perimetry, microperimetry and rarebit perimetry in patients suffering from dry age related macular degeneration]. / Ocena funkcji plamki za pomoca perymetrii statycznej, mikroperymetrii oraz perymetrii "rarebit" u chorych z postacia sucha AMD.

PURPOSE: To compare the visual field results obtained by static perimetry, microperimetry and rabbit perimetry in patients suffering from dry age related macular degeneration (AMD). MATERIAL AND METHODS: Fifteen eyes with dry AMD (hard or soft macula drusen and RPE disorders) were enrolled into the study. Static perimetry was performed using M2 macula program included in Octopus 101 instrument. Microperimetry was performed using macula program (14-2 threshold, 10dB) within 10 degrees of the central visual field. The fovea program within 4 degrees was used while performing rarebit perimetry. RESULTS: The mean sensitivity was significantly lower (p<0.001) during microperimetry (13.5 dB) comparing to static perimetry (26.7 dB). The mean deviation was significantly higher (p<0.001) during microperimetry (-6.32 dB) comparing to static perimetry (-3.11 dB). The fixation was unstable in 47% and eccentric in 40% while performing microperimetry. The median of the "mean hit rate" in rarebit perimetry was 90% (range 40-100%). The mean examination duration was 6.5 min. in static perimetry, 10.6 min. in microperimetry and 5,5 min. in rarebit perimetry (p<0.001). Sensitivity was 30%, 53% and 93% respectively. CONCLUSIONS: The visual field defects obtained by microperimetry were more pronounced than those obtained by static perimetry. Microperimetry was the most sensitive procedure although the most time-consuming. Microperimetry enables the control of the fixation position and stability, that is not possible using the remaining methods. Rarebit perimetry revealed slight reduction of the integrity of neural architecture of the retina. Microperimetry and rarebit perimetry provide more information in regard to the visual function than static perimetry, thus are the valuable method in the diagnosis of dry AMD.

[Intravitreal bevacizumab (Avastin) iniections for neovascular age-related macular degeneration (AMD)--preliminary results]. / Doszklistkowe iniekcje bevacizumabu (Avastin) w leczeniu wysiekowej formy zwyrodnienia plamki zwiazanego z wiekiem (AMD)--doniesienie wstepne.

PURPOSE: To evaluate effects of intravitreal bevacizumab on visual acuity and angiographic lesions characteristics in patients with neovascular AMD and to report safety of such treatement. MATERIAL AND METHODS: 32 patients with confirmed choroidal neovascularisation (CNV) and AMD were treated. Patients received 2 intravitreal bevacizumabu (1.25 mg) iniections on 1-3 months basis. Control ophthalmic evaluations included visual acuity measurements with EDTRS charts, intraocular pressure measurements, complete ophthalmic examination in slit lamp, fluorescein angiography and blond pressure measurements. RESULTS: In 18 patients (56%) visual acuity improvement at a mean 3 lines on EDTRS charts was observed, in 9 patients (28%) visual acuity did not change and in 5 (16%) cases decreased visual acuity (about 1,5 lines in EDTRS charts) was noted. Mean follow-up period was 3 months after second injection. In most study eyes fluorescein angiography revealed a marked reduction in leakage from CNV. Apart from one case with endophthalmitis symptoms after second bevacizumab injection, we did not observed systemic or ocular adverse effects of the applied treatment. CONCLUSIONS: Short-term results suggest that intravitreal bevacizumab is relatively safe form of AMD treatement and is associated with improvement in visual acuity and reduction in angiographic leakage in most patients with neovascular AMD. Further evaluation of the long-term effectiveness of this treatment is warranted.

Alterations of amino acids and glutamate transport in the DBA/2J mouse retina; possible clues to degeneration.

BACKGROUND: The DBA/2J mouse spontaneously develops ocular hypertension and time-dependent progressive retinal ganglion cell (RGC) loss. This study examines changes in amino acid levels in the vitreous, and changes in the expression of retinal glutamate transporters and receptors that occur during the progression of this pathology. METHODS: Retinas were obtained from DBA/2J mice at ages 3, 6 and 11 months. C57BL/6 mice were used as age-matched controls. Vitreal amino acid content was measured with HPLC. Western blotting and immunohistochemistry were performed using specific antibodies against the glutamate transporters (GLAST, GLT-1v, EAAC-1) and glutamate receptors, particularly NMDA (NR1, NR2A, NR2B) and AMPA (GluR1, GluR2/3, GluR4) receptors. RESULTS: HPLC showed retinal concentrations of glutamate, glutamine, glycine, alanine, lysine, serine, and arginine to be significantly higher in DBA/2J mice at 11 months of age compared to age-matched controls. Western Blots revealed a moderate decrease of GLAST and GLT-1v expression in DBA/2J mice at 6 and 11 months as compared to age-matched controls while there was no change in EAAC1. Immunohistochemically, no changes in expression of NMDA and AMPA receptors were seen. CONCLUSION: Alterations of amino acid content and enhanced glutamate neurotransmission might be involved in the pathogenesis of retinal neurodegeneration in the DBA/ 2J mouse model of ocular hypertension. Moreover, these mice provide an animal model for studying excitotoxic retinal damage.

Administration of novel dyes for intraocular surgery: an in vivo toxicity animal study.

PURPOSE: To investigate the effect of intravitreal injections of new vital dyes on the retina, the retinal pigment epithelium (RPE) and the choroid in an in vivo rat model. METHODS: Rats were injected intravitreally with four dyes: light-green SF yellowish (LGSF), copper(II)phthalocyanine-tetrasulfonic acid (E68), bromphenol blue (BPB), and Chicago blue (CB) dissolved in physiologic saline solution (PSS) at concentrations of 0.5% and 0.02%. PSS served as the control. Additional animals were treated with single injections of 0.5%, 0.02%, 0.002%, and 0.0002% ICG or 0.002% E68 into one eye. Adverse effects on anterior and posterior segments were evaluated by slit lamp biomicroscopy and ophthalmoscopy. Retinal toxicity was assessed by histology and retinal ganglion cell (RGC) quantification 7 days after dye administration. RESULTS: Eyes treated with 0.5% E68, 0.5% ICG, or 0.5% CB showed discrete staining of both cornea and lens not seen at lower concentrations or with other dyes. Histology revealed dose-dependent reactions after E68 administration. ICG 0.5% induced significant thinning of inner retinal layers compared with PSS. ICG 0.02% caused focal degenerative changes of the outer retina in three of seven eyes, whereas 0.002% and 0.0002% ICG did not. CB led to heterogeneous morphologic alterations. BPB- or LGSF-treated eyes showed normal retinal morphology. ICG at all tested concentrations induced significant RGC loss, as did E68 at 0.5% but not at lower concentrations. CONCLUSIONS: BPB or LGSF produced no significantly detectable toxic effects on the retina in vivo. The safety of these new dyes must be established in other models and/or in preclinical studies before the clinical use of any of these dyes.

Citicoline and lithium rescue retinal ganglion cells following partial optic nerve crush in the rat.

Citicoline and lithium (Li(-)) have been shown to support retinal ganglion cell (RGC) survival and axon regeneration in vitro. Optic nerve crush (ONC) is a model of both brain axonal injury and certain aspects of the glaucomatous degeneration of RGC. We have used this model to quantify protection offered to RGC by these drugs and to determine whether their effects are mediated by enhanced expression of the antiapoptotic protein Bcl-2. Adult rats (6-12 per group) were subjected to ONC accompanied by a contralateral sham operation. Animals were treated intraperitoneally with either vehicle, citicoline sodium (1g/kg daily for up to 7 days and 300 mg/kg daily afterwards), lithium chloride (30 mg/kg daily), or both drugs combined. Fluorogold was injected bilaterally into superior colliculi 1, 5 or 19 days after ONC. Labeled cells were counted under a fluorescence microscope 2 days after tracer injection. In a separate set of experiments the effects of treatments on expression of Bcl-2 in retinas were evaluated by immunohistochemistry. In vehicle-treated animals there was a progressive decrease of RGC density after crush. This decrease was attenuated in citicoline-treated animals 1 week and 3 weeks after the crush. In the lithium-treated group protection was even more pronounced. In animals treated with both drugs RGC protection was similar to that achieved by lithium alone. Bcl-2 immunoreactivity was seen predominantly in retinal ganglion cells. Its increase was recorded in the lithium and citicoline group as well as in animals treated with the combination of both drugs. Both citicoline and lithium protect RGC and their axons in vivo against delayed degeneration triggered by the ONC. Retinoprotective action of both drugs may involve an increase in Bcl-2 expression.

[Efficacy of intravitreal triamcinolone acetonide in the visualization of the vitreous during pars plana vitrectomy]. / Zastosowanie octanu triamcinolonu w celu wizualizacji ciala szklistego w przebiegu witrektomii pars plana.

PURPOSE: To retrospectively evaluate the efficacy of triamcinolone acetonide (TA) injection during pars plana vitrectomy, to facilitate the visualization of the internal limiting membrane (ILM), residual vitreous cortex (RVC) and preretinal membranes. MATERIAL AND METHODS: Pars plana vitrectomy was performed in 164 patients due to rhematogenous retinal detachment, vitreous hemorrhage, macular hole, lens luxation, endophthalmitis, intraocular foreign body, malignant glaucoma, preretinal membrane and proliferative diabetic retinopathy. After surgical separation of the posterior vitreous and removal of any visible epiretinal membrane, TA suspension was injected over the posterior pole into the vitreous cavity. Then, visualized RVC was removed and ILM peeling was performed. RESULTS: Upon intravitreal injection of TA, vitreous and ILM could be visualized by numerous particles of TA dispersed as white specks. RVC and ILM were completely removed in all patients. No complications related to the use of TA were encountered, even after complex procedures such as, vitrectomy combined with scleral buckling or phakoemulsification surgery in a long term follow up. CONCLUSIONS: Intraoperative visualization of RVC and ILM with intravitreal TA was found to be a useful adjunct to pars plana vitrectomy. This technique may facilitate both removal of epiretinal membrane and separation of vitreous, especially in patients with undetached vitreous.

Retinopathy of prematurity: A refresher for obstetricians.

Advances in neonatal care improved the survival of many preterm infants, but also increased the incidence of retinopathy of prematurity (ROP). Numerous risk factors have been associated with the development of ROP, the most important of which are: low birth weight, early gestational age at delivery, and duration of oxygen therapy. Screening premature infants is a critical factor for any prevention and treatment protocol. The Retinopathy of Prematurity Subcommittee of the American Academy of Ophthalmology (AAO) and the American Association for Pediatric Ophthalmology and Strabismus (AAPOS) have developed guidelines that assist practicing physicians in managing children with ROP. A portion of these guidelines is particularly applicable to practicing obstetricians who, along with the pediatricians, are often the primary physicians for the affected families. The aim of this paper is to present a comprehensive overview of the epidemiology, etiology, manifestations, prevention, and management of ROP. In the last section, the authors present guidelines for practicing obstetricians that incorporate the most recent recommendations of ophthalmic and pediatric thought leaders.

[The Vogt-Koyanagi-Harada syndrome in 11-year-old girl]. / Zespól Vogta-Koyanagi-Harady u 11-letniej dziewczynki.

PURPOSE: The Vogt-Koyanagi-Harada syndrome is an idiopathic, multisystem disorder which typically affects pigmented individuals between 20-50 years old. The typical symptoms include granulomatous panuveitis with characteristic extraocular dermato-neurological manifestations. The case of VKH syndrome affecting Polish girl is very rare. CASE REPORT: A 11-year-old girl had an important decrease of visual acuity in both eyes (RE 0.1 and LE 0.2), with the presence of bullous serous retinal detachment in both eyes. Besides, in right eye some keratic precipitates were noted. The routine laboratory evaluation failed to provide a diagnosis. The patient was treated with an intravenous bolus of corticosteroid therapy and then, high dose of oral prednisone. RESULTS: After administered therapy the visual acuity improved rapidly in both eyes (RE 1.0 and LR 0.9), and the exudative retinal detachment resolved. After 5 months, the patient's examination showed a sunset glow fundus with several whitish rounded lesions in peripheral fundus, which is typical for the VKH syndrome. CONCLUSIONS: The VKH syndrome rarely affects young children, so the diagnosis may be difficult in the absence of the typical extraocular manifestations. In such atypical cases the diagnosis is based on the clinical evolution of the disease.