Total RNA quality in boar spermatozoa with different freezability.

In this study the quality of total RNA, isolated from fresh spermatozoa, was compared between boars with good and poor semen freezability (GSF and PSF, respectively). Semen from 3 boars with GSF exhibited significantly higher total motility, mitochondrial function, plasma membrane integrity and reduced lipid peroxidation compared with 3 boars with PSF after cryo- preservation. There were variations in the quality of RNA isolated from spermatozoa of boars with GSF and PSF. Boars with GSF exhibited mainly full-length, intact RNA, whereas substantial amounts of degraded RNA were detected in spermatozoa from boars with PSF. Further under- standing of the biological relevance of RNAs in sperm function is critical to improve the freezabil- ity of boar semen.

Boar variability in sperm cryo-tolerance after cooling of semen in different long-term extenders at various temperatures.

This study investigated individual boar variability in the quality of pre-freeze (PF) and post-thaw (PT) semen cooled in different long-term (LT) extenders and for different holding times (HT). Sperm rich fractions were diluted with Androhep® Plus (AHP), Androstar® Plus (ASP), Safecell® Plus (SCP) and TRIXcell® Plus (TCP) extenders, stored for 2h at 17°C (HT 1) and additionally for 24h at 10°C (HT 2) and the samples were subsequently evaluated and frozen. Besides the analysis of CASA sperm variables, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI), normal apical ridge (NAR) acrosome integrity, and viability (YO-PRO-1-/PI-) of sperm were assessed in the PF and PT semen. Results indicated that boar, extender and HT group affected the sperm quality characteristics. There were great variations in PMOT and the sperm motion patterns of the PF semen among the boars. Differences in the HT groups of the PF semen, with respect to the sperm membrane integrity, were less marked among the boars. Consistent variations in TMOT and PMOT in the PT semen were observed among the boars, being greater in the HT 2 group. Most of the CASA-analyzed sperm motion patterns were greater in the HT 2 group of the PT semen. Furthermore, sperm MMP, PMI and viability were greater in the HT 2 group of the PT semen in most of the boars, while consistent differences were observed among the boars for sperm NAR acrosome integrity in either HT group. The significant effect of the cryopreservation process on the sperm membrane proteome was evident from the number of protein bands, detected in the electrophoretic profiles of sperm of the HT 1 and HT 2 groups. The electrophoretic profiles of the PF and PT semen among boars with poor and good semen freezability, however, differed with respect to the abundance and types of sperm membrane-associated proteins. The overall results of this study provided evidence that there are differences among boars in response to the different cooling regimens, and that cooling of extended semen for a 24-h period at 10°C modulated the functions of sperm in an extender-dependent manner, rendering the cells less susceptible to cryo-induced damage. It is suggested that the findings of this study have the potential to improve the technology of boar semen cryopreservation.

The benefits of cooling boar semen in long-term extenders prior to cryopreservation on sperm quality characteristics.

This study investigated the effects of long-term extenders on post-thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm-rich fractions, collected from five boars, were diluted in Androhep(®) Plus (AHP), Androstar(®) Plus (ASP), Safecell(®) Plus and TRIXcell(®) Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre-freeze and frozen-thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic-like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing-thawing. Differences in the pre-freeze semen were more marked in the sperm motion patterns between the HTs. Pre-freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO-PRO-1(-) /PI(-) ) among the extenders. Post-thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP- and ASP-extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing-thawing. In most of the extenders, the incidence of frozen-thawed spermatozoa with apoptotic-like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long-term preservation extenders modulates post-thaw sperm quality characteristics in an extender-dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.

ALA-induced photodynamic effect on vitality, apoptosis, and secretion of vascular endothelial growth factor (VEGF) by colon cancer cells in normoxic environment in vitro.

BACKGROUND: Cancer therapy is often based on combination of conventional methods of cancer treatment with immunotherapy. Photodynamic therapy (PDT) is one of the immunomodulating methods used in oncology. We examined how PDT influences the secretory activity of colon cancer cells in vitro, especially the secretion of vascular endothelial growth factor (VEGF) in aerobic conditions. METHODS: We used two cancer cell lines with different malignancy potentials: a metastatic SW620 line and a non-metastatic SW480 line. In the first stage of the experiment, we exposed each cell line to three different concentrations of photosensitizer's precursor: 5-aminolevulinic acid (ALA) and varying levels of light radiation, after which we assessed cell viability and apoptosis induction in these lines, using the MTT and LDH assays. Then, we determined the secretion of VEGF by these cells in aerobic conditions and under the ALA-PDT parameters at which cells presented the highest viability. RESULTS: Photodynamic treatment with ALA did not influence on VEGF secretion by the non-metastatic SW480 cells, but caused a decrease in VEGF secretion by the metastatic SW 620 cell line by 29% (p<0.05). SW 620 cell line secreted more actively VEGF than the SW480 cells, both before and after photo dynamic therapy (p<0.05). CONCLUSION: The outcome of this in vitro study presented a beneficial effect of ALA-PDT, resulting in a decrease of VEGF secretion in the more malignant SW620 cell lines. Further studies should be considered to confirm the clinical relevance of this finding.

Vascular-targeted photodynamic therapy in the treatment of neovascular age-related macular degeneration: Clinical perspectives.

Vascular targeted photodynamic therapy (VTP), with use of verteporfin as a photosensitizer is one of the few therapies, which has been shown to effectively slow the progression of the "wet" form of age-related macular degeneration (AMD), and even to stabilize visual acuity over many years. Although, due to considerable advance of AMD treatment, it is currently not recommended in monotherapy of AMD, however, its combination with steroids and anti-angiogenic biologic drugs may reveal high therapeutic potential in the treatment of neovascular AMD. The future of VTP as a method of AMD treatment is development of new selective and targeted photosensitizer and combination of this method with other therapeutic strategies targeting cellular structures or pathways involved in AMD progression.

Prostasomes of canine seminal plasma - zinc-binding ability and effects on motility characteristics and plasma membrane integrity of spermatozoa.

Prostasomes are small lipid membrane-confined vesicles that are involved in various fertilization-related processes. The aim of this study was to demonstrate canine seminal plasma prostasomes' ability to bind zinc ions, as well as examining their effects on sperm motility characteristics and plasma membrane integrity during cold storage. Ejaculates, collected from five cross-bred dogs (n = 50), were subjected to ultracentrifugation followed by gel filtration (GF) on a Superose 6 column. Prostasomes appeared as a single fraction in the elution profile. Transmission electron microscopy (TEM) analysis of canine prostasomes revealed the presence of membrane vesicles with diameters ranging from 20.3 to 301 nm. The zinc-affinity chromatography on a Chelating Sepharose Fast Flow - Zn(2 +) showed that from 93 to 100% of the prostasome proteins bind zinc ions (P(+) Zn). SDS-PAGE revealed that canine P(+) Zn comprised four protein bands, with low molecular weights (10.2-12 kDa). We have also shown a positive effect of prostasomes (p < 0.05), especially variant B (2% of total seminal plasma protein) on canine sperm motility parameters after 2 h storage at 5°C (TMOT%, 44.75 ± 5.18) and PMOT%, 12.42 ± 1.59) and VAP, VSL, VCL, when compared with Control (TMOT%, 7.30 ± 1.41 and PMOT%, 1.70 ± 0.42). Higher percentage of spermatozoa with intact plasma membrane (SYBR/PI dual staining) and intact acrosome (Giemsa stained), after 2 h storage at 5°C, was showed, in variant A (1.5% of total seminal plasma protein) and B, when compared with Control and variant C (2.5% of total seminal plasma protein). The prostasomes' effect on motility and plasma membrane integrity of canine cold-stored spermatozoa may be related to their ability to bind zinc ions and regulate their availability to the sperm.

Individual and seasonal variations in the quality of fractionated boar ejaculates.

Reproductive seasonality has been shown to affect the quality of boar semen. In this study, effects of seasonal variations in the characteristics of spermatozoa and seminal plasma (SP) of fractioned ejaculates from individual boars have been investigated. Fractionated ejaculates, designated as fraction 1 (F1), fraction 2 (F2), and fraction 3 (F3), were collected from five mature boars during the autumn-winter (October through March) and spring-summer periods (April through September). A total of 10 fractionated ejaculates (F1, F2, and F3) were collected from each boar within each seasonal period. Assessments of the sperm quality characteristics included computer-assisted sperm analysis motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity, normal apical ridge acrosomes, and DNA fragmentation. Besides SDS-PAGE and densitometric analyses of the SP proteins, the antiperoxidant activity was monitored. There were marked differences in the sperm quality characteristics among the boars, except for sperm MMP. Distinct seasonal differences (P < 0.05) were observed in the ejaculate volume of F3 during the autumn-winter and spring-summer periods (107.78 ± 5.45 and 87.80 ± 4.75 mL, respectively). Significantly higher (P < 0.05) sperm concentration and the total number of spermatozoa in the fraction were observed during the autumn-winter period. Seasonal effects in MMP and plasma membrane integrity were manifested in significantly higher (P < 0.05) percentages of spermatozoa with functional mitochondria and intact plasma membrane during the autumn-winter period. However, the seasonal effects were less marked in either sperm normal apical ridge acrosomes or sperm DNA fragmentation. Sodium dodecyl sulfate-PAGE and densitometric analyses revealed marked variations in the protein composition of the SP profiles among the boars, regardless of the ejaculate fraction and seasonal period. Distinct seasonal variations, observed in the SDS-PAGE profiles, were associated with an abundance of protein fractions of low-molecular and high-molecular weight components, particularly during the autumn-winter period. There were wide variations in antiperoxidant activity in the SP among the boars, being significantly higher in the autumn-winter period, irrespective of the ejaculate fraction. It can be suggested that marked deterioration of the quality of fractionated ejaculates during the spring-summer period was probably caused by impaired reproductive function in the boar.