Short‐Term Antiandrogen Flutamide Treatment Causes Structural Alterations in Somatic Cells Associated with Premature Detachment of Spermatids in the Testis of Pubertal and Adult Guinea Pigs

In spite of widespread application of flutamide in the endocrine therapies of young and adult patients, the side effects of this antiandrogen on spermatogenesis and germ‐cell morphology remain unclear. This study evaluates the short‐term androgen blockage effect induced by the administration of flutamide to the testes of pubertal (30‐day old) and adult (65‐ and 135‐day old) guinea pigs, with an emphasis on ultrastructural alterations of main cell types. The testes removed after 10 days of treatment with either a non‐steroidal antiandrogen, flutamide (10 mg/kg of body weight) or a pharmacological vehicle alone were processed for histological, quantitative and ultrastructural analysis. In pubertal animals, flutamide androgenic blockage induces spermatogonial differentiation and accelerates testes maturation, causing degeneration and detachment of primary spermatocytes and round spermatids, which are subsequently found in great quantities in the epididymis caput. In post‐pubertal and adult guinea pigs, in addition to causing germ‐cell degeneration, especially in primary spermatocytes, and leading to the premature detachment of spherical spermatids, the antiandrogen treatment increased the relative volume of Leydig cells. In addition, ultrastructural evaluation indicated that irrespective of age antiandrogen treatment causes an increase in frequency of organelles involved with steroid hormone synthesis in the Leydig cells and a dramatic accumulation of myelin figures in their cytoplasm and, to a larger degree, in Sertoli cells. In conclusion, the transient exposition of the guinea pigs to flutamide, at all postnatal ages causes some degenerative lesions including severe premature detachment of spermatids and accumulation of myelin bodies in Leydig and Sertoli cells, compromising, at least temporarily, the spermatogenesis.     

Novel biological properties of Oenothera paradoxa defatted seed extracts: effects on metallopeptidase activity

In this study, for the first time, we used the in vitro metallopeptidase model for the identification of a potential novel activity of defatted evening primrose seed extracts. Prepared extracts of different polarity (aqueous, 60% ethanolic, isopropanolic, and 30% isopropanolic) at concentrations of 1.5-100 microg/mL exhibited a significant and dose dependent inhibition of three tested enzymes. The 50% inhibition of enzymes activity showed that aminopeptidase N (APN) was the enzyme affected to the greatest extent with IC50 at the level of 2.8 microg/mL and 2.9 microg/mL for aqueous and 30% isopropanolic extracts, respectively. The activity of neutral endopeptidase (NEP) was quite strongly inhibited by the extracts as well. The HPLC-DAD analysis and bioguided fractionation led to the identification of four active compounds: (-)-epicatechin gallate, proanthocyanidin B3, oenothein B, and penta-O-galloyl-beta-D-glucose (PGG). Oenothein B has been shown previously to inhibit metallopeptidases. The three other compounds are known to inhibit angiotensin-converting enzyme (ACE), but they have not been previously reported to inhibit the NEP and APN activity. PGG and procyanidins with different degrees of polymerization, as the dominating compounds in O. paradoxa seeds, seemed to play a role in the crude extract activity.     

Cryopreservation of Zona‐Free Cloned Buffalo (Bubalus Bubalis) Embryos: Slow Freezing vs Open‐Pulled Straw Vitrification

This study was carried out to compare the post‐thaw cryosurvival rate and the level of apoptosis in vitro produced zona‐free cloned buffalo blastocysts subjected to slow freezing or vitrification in open‐pulled straws (OPS). Zona‐free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re‐expansion rate following post‐thaw culture for 22–24 h. The post‐thaw re‐expansion rate was significantly (p < 0.05) higher following vitrification in OPS (71.2 ± 2.3%) compared with that after slow freezing (41.6 ± 4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen‐thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non‐cryopreserved cloned embryos (422.6 ± 67.8) and those cryopreserved by slow freezing (376.4 ± 29.3) or vitrification in OPS (422.8 ± 36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8 ± 2.0) or OPS vitrification (13.3 ± 1.8), was significantly (p < 0.05) higher than that for the control non‐cryopreserved cloned embryos (3.4 ± 0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona‐free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.     

Comparison of intradermal testing and serum testing for allergen-specific IgE using monoclonal IgE antibodies in 84 atopic dogs

OBJECTIVE: To compare an ELISA measuring serum allergen-specific IgE with intradermal skin testing in canine atopic dermatitis. PROCEDURE: Eighty-four dogs with the clinical diagnosis of atopic dermatitis underwent intradermal skin testing and serum testing for allergen-specific IgE. Tests were performed in a blinded fashion. Positive reactions were compared and the sensitivity and specificity of the serum test (using intradermal skin test as the standard) were determined overall and for individual allergen groups (grass pollens, weed pollens, tree pollens, house dust mites and fleas). RESULTS: The sensitivity of the ELISA overall was 90.4%. Evaluating the individual allergen groups, the sensitivity for dust mite hypersensitivity was 95.1%, for fleas 85.4%, for tree pollens 84.3%, for grass pollens 95.1% and for weed pollens 96.4%. The specificity was 91.6% overall, for dust mites 96.3%, for fleas 92.7%, for tree pollens 95.2%, for grass pollens 94% and for weed pollens 80.7%. CONCLUSION: The evaluated ELISA seemed reliable for the diagnosis of atopy in practice and can be recommended as a screening test prior to intradermal skin testing or for use in dogs when immunotherapy is not a therapeutic option.     

Molecular evidence in support of recent migration of a powdery mildew fungus on Syringa spp. into Europe from East Asia

A preliminary analysis demonstrated that the powdery mildew fungus infecting Syringa spp. (lilacs) in different parts of the world is divided into two groups (S-type and K-type) based on the nucleotide sequences of the rDNA ITS regions. In spite of the marked genetic differences (only c . 94% similarity between ITS types), fungi belonging to these two ITS groups are difficult to distinguish based on morphological characteristics. To determine their geographical distribution, ITS haplotypes were determined for a total of 139 powdery mildew specimens collected in Asia, Europe and North and South America between 1977 and 2005. Curiously, until 1990, only the S-type was found in Europe, whilst the K-type prevailed in East Asia. The first European specimen belonging to the K-type was collected in Ukraine in 1991. Other European K-type samples were collected in Lithuania and Switzerland in 2000, and in other countries after 2002. The incidence of the S-type decreased rapidly in Europe after the 1990s. This result strongly suggests that the K-type was introduced to Europe from East Asia in the 1990s and expanded from Eastern Europe westward, replacing the S-type present on the European lilacs. The K-type produces abundant chasmothecia (sexual fruiting bodies), whereas chasmothecia on the S-type are rare in Europe and East Asia. It is likely that the recent abundant production of chasmothecia on Syringa spp. in Central Europe is explained by the migration of the K-type to Europe.     

Morphological and RAPD analysis of poplar trees of anther culture origin

Our objectives were to improve the rate of haploid plant regeneration through increasing the rate of callus initiation on the anthers and sustaining shoot regeneration frequency, and to analyze the field population of anther culture origin by morphological and molecular methods. Regarding the callus initiation, the most responsive clones were ‘N-90’(59%) in P. nigra and ‘D-29’ (75%) in P. deltoides. The rate of shoot regeneration and number of shoots/calli ranged from 4%–79% and 1–9, respectively. From the 208 rooted plants 8 haploid, 179 diploid, 4 tetraploid and 17 aneuploid plants were found. In the field population, the haploid plants could be easily identified by their retarded development and morphological characteristics (size and shape of the leaves, strong branching, etc). Several diploid plants showed depressed developmental and morphological traits similar to the haploid ones. Three traits (growth rate, leaf blade length and shape of leaf base) of the 6 different morphological characteristics measured were in correlation with the ploidy level within the poplar field population. Six primers of the 48 primers tested were able to detect polymorphism among the field plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Activation of Zona‐Free Buffalo (Bubalus bubalis) Oocytes by Chemical or Electrical stimulation,and Subsequent Parthenogenetic Embryo Development

Parthenogenetic activation using zona‐free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona‐free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6‐dimethylaminopurine (6‐DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona‐free oocytes when compared to zona‐intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona‐free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6‐DMAP resulted in the highest blastocyst rate.