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DNA integrity of fresh and frozen canine epididymal spermatozoa
No full textThe aims of this study were to evaluate: (1) the effect of cryopreservation on DNA fragmentation of canine epididymal spermatozoa, and (2) the potential protective effect of melatonin on post-thaw sperm quality (motility, morphology, acrosomal and DNA integrity). Epididymal spermatozoa were collected after orchiectomy of ten dogs. Sperm samples were frozen in the presence or absence of melatonin (1 mM). DNA fragmentation index (percentage of spermatozoa with fragmented DNA) was similar in fresh samples (3.3 ± 3.6) and samples frozen with (4.2 ± 3.8) or without (3.6 ± 3.7) melatonin. Sperm motility was significantly (p < 0.0001) higher in fresh compared to frozen samples. The presence of melatonin in the freezing extender did not affect the sperm motility. Proportions of spermatozoa with normal morphology were similar in fresh and frozen samples, irrespective of the presence of melatonin in the extender. Acrosome integrity was significantly decreased (p < 0.01) by cryopreservation, and melatonin did not exert any beneficial effects. In conclusion, DNA fragmentation of canine epididymal spermatozoa was not affected by the freezing procedure, and the presence of melatonin did not preserve motility and acrosome integrity which were adversely affected by cryopreservation. The evaluation of DNA status of thawed gametes is particularly relevant for epididymal spermatozoa since these spermatozoa are usually stored and used in assisted reproductive techniques
Quality of canine spermatozoa retrieved by percutaneous epididymal sperm aspiration
No full textOBJECTIVES : To investigate the feasibility of percutaneous epididymal sperm aspiration in dogs and
whether it might provide a population of epididymal spermatozoa similar to the population that can be
obtained by processing isolated epididymis caudae.
METHODS : Concentration and total sperm number, motility, morphology and acrosomal integrity of sper-matozoa retrieved by percutaneous epididymal sperm aspiration, in vitro aspiration and mincing of the
cauda of the epididymis were compared.
RESULTS: Percutaneous epididymal sperm aspiration is a feasible procedure to retrieve a population of
spermatozoa in dogs. Quality is similar to that of spermatozoa collected in vitro, although a wide varia-tion amongst animals was observed.
CLINICAL S IGNIFICANCE : In case of ejaculation failure due to pathological conditions in dogs, the collection
of spermatozoa from the cauda of the epididymis could be an option for providing gametes for assisted
reproductive technologies. Percutaneous epididymal sperm aspiration can be used in dogs with com-promised reproductive performance, in which orchiectomy cannot be performed for medical or owner
reasons. Further studies aimed to investigate whether the percutaneous epididymal sperm aspiration
technique might be feasible for repeated semen collection and to accurately evaluate side effects are
required
Morphological and acrosomal changes of canine spermatozoa during epididymal transit
Get PDFBackground: During epididymal transit, functional and structural modifications leading to full maturation enable male gametes to reach, recognize and fertilize the oocytes. In dogs, little is known on the modifications of spermatozoa during the passage in the epididymis. The aim of this study was to describe the motility, morphology and acrosomal patterns of canine spermatozoa retrieved from the epididymis caput, corpus and cauda.Results: After the dilution required for the collection of epididymal content, sperm motility was significantly higher(P<0.0001) in the cauda compared to corpus and caput.Proportions of spermatozoa with normal morphology were significantly higher in corpus (P =0.02) and cauda(P<0.0001) compared to caput. Overall morphological abnormalities of the head and neck/midpiece were similar in the three different epididymal regions. A significantly increased prevalence of tail defects, mainly represented by single bent tails, was observed in the corpus compared to caput (P<0.0001) and cauda (P=0.006).Numbers of immature sperm with cytoplasmic droplets decreased from the proximal to the distal region of the epididymis. Particularly, proximal cytoplasmic droplets were more frequently found in spermatozoa collected from the caput epididymis than in the corpus (P<0.0001) and in the cauda (P<0.0001), whereas the occurrence of distal cytoplasmic droplets was higher in the corpus than in the caput (P=0.0003) and in the cauda (P<0.05).Significantly higher proportions of spermatozoa with intact acrosomes were retrieved from the cauda epididymis than from the caput (P=0.03) and the corpus (P =0.008). This difference was mainly due to a lower proportion of spermatozoa with abnormal acrosomes (mainly swollen acrosomes) rather than with absent acrosomes. Conclusions: Canine spermatozoa undergo several modifications in the epididymis. The acquisition of progressive motility, migration of the cytoplasmic droplet and acrosomal reshaping lead to mature spermatozoa which are then stored in the cauda epididymis. From this site, spermatozoa can be retrieved and used in assisted reproductive techniques as a valuable tool for propagating genetic traits of high value individuals that dies accidentally or undergoes orchiectomy for medical purposes. Further investigations should be also focused on the potential use of spermatozoa recovered from other epididymal regions
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Sperm ubiquitination in epididymal feline semen
No full textUbiquitin is a 8.5-kDa peptide that tags other proteins for proteasomal degradation. It has been proposed that ubiquitination might be responsible for the elimination of defective spermatozoa during transit through the epididymis in humans and cattle, but its exact biological function in seminal plasma has not yet been clarified. In the domestic cat (Felis catus), the percentage of immature, unviable, and abnormal spermatozoa decreases during the epididymal transit, indicating the existence of a mechanism that removes defective spermatozoa. Magnetic cell separation techniques, based on the use of magnetic beads coated with anti-ubiquitin antibodies, may allow the selective capture of ubiquitinated spermatozoa from semen, thus contributing to the identification of a potential correlation between semen quality and ubiquitination process. Moreover, the selective identification of all the ubiquitinated proteins in different epididymal regions could give a better understanding of the ubiquitin role in feline sperm maturation. The aims of this study were as follows: (1) to verify the possibility of separating ubiquitinated spermatozoa with magnetic ubiquitin beads and identify the morphological and acrosomal differences between whole sample and unbound gametes, (2) to characterize all the ubiquitinated proteins in spermatozoa retrieved in the three epididymal regions by a proteomic approach. The data indicated the presence of ubiquitinated proteins in cat epididymal semen. However, a correlation between abnormal and ubiquitinated spermatozoa has not been found, and ubiquitin cannot be considered as a biomarker of quality of epididymal feline spermatozoa. To the author's knowledge, this is the first identification of all the ubiquitinated proteins of cat spermatozoa collected from different epididymal regions. The proteomic pattern allows a further characterization of cat epididymal semen and represents a contribute to a better understanding of the ubiquitin role in feline sperm maturation
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Sperm ubiquitination in epididymal feline semen
No full textINTRODUCTION. Ubiquitin is a 8.5 kDa peptide that tags other proteins for proteasomal degradation and it is also involved in the regulation of protein function. Ubiquitin has been discovered as a normal component of human blood, ovarian follicular fluid and seminal plasma (1). Ubiquitination might be responsible of the elimination of defective spermatozoa during transit through epididymis in humans and cattle (2, 3). Results indicated that the increase of sperm ubiquitin was inversely associated with spermatic concentration, motility and normal morphology indicating that ubiquitination could be considered as a biomarker of poor semen quality (3). Conversely, other authors (4) found a positive correlation between sperm ubiquitin and good semen parameters suggesting a different role for sperm ubiquitination. These conflicting data indicate that the exact biological function of this peptide in seminal plasma has not yet been clarified. In the domestic cat (Felis catus) no positive or negative correlations between semen quality and ubiquitination have been observed, although no definitive conclusions about its role have been drawn (5). Magnetic cell separation techniques, based on the use of antibodies or proteins-coated magnetic beads as magnetic ubiquitin beads, may allow the removal of ubiquitinated spermatozoa from the sample contributing to the identification of a potential correlation between ubiquitin and abnormal spermatozoa.
OBJECTIVES AND METHODS. The present study was designed to investigate whether ubiquitin could be considered a biomarker of quality of epididymal feline semen. Morphology and acrosomal integrity of spermatozoa were correlated with ubiquitination of the protein patterns in semen samples treated with magnetic ubiquitin beads. Moreover, the modification of the two major cytoskeletal proteins (actin and tubulin), and of prohibitin, a protein involved in cell cycle which is ubiquitinated in bovine semen (6), was evaluated in details to investigate the possible correlation of sperm morphological alterations and proteins ubiquitination.
Semen samples from 10 healthy and sexually mature cats were collected by squeezing isolated caudae epididymis after routinary orchiectomy. Samples were divided into two aliquots. Magnetic ubiquitin beads (Li Starfish S.r.l., Cernusco S/N, Milan, Italy) were added to one aliquot at the final concentration of 5 particles/spermatozoa. The suspension in the tube was gently mixed for 15 min before placing it on an external laboratory magnet for 15 min. The sample treated with beads (S+B) was collected while the tube was still in the magnetic field, whereas the “ubiquitinated” spermatozoa bound to the beads remained attached to the wall of the tube as long as the magnet was in place. The second semen aliquot was used as a control and was treated under the same conditions except for the presence of the beads (S-B). Before and after the treatment, sperm cell concentration was determined with a Bürker chamber and semen morphology was assessed following staining with Bengal Rose and Victoria Blue B on at least 100 spermatozoa per slide. Acrosome integrity was analyzed by staining with Peanut agglutinin (PNA) conjugated with a fluorescein isothiocyanate (FITC) and propidium iodide (PI). Mean±SD of sperm characteristics were analyzed by Student’s t-test (p<0.05).
Assessment of the ubiquitination of sperm proteins was carried out by Western Blot analysis using specific antibodies, namely anti-ubiquitin, anti-actin, anti-tubulin and anti-prohibitin. Each sample was solubilized in lysis buffer (50mM Tris HCl pH 8.0) and quantified for its protein content by the Bradford method. Then the samples were separated by sodium dodecyl sulphate gel electrophoresis upon sonication and centrifugation. Following blotting on polivinilidenfluoruro membrane, ubiquitinated proteins, actin, tubulin and prohibitin were detected by a chemiluminescence immunoassay.
RESULTS. Proportions of morphologically normal spermatozoa were similar in samples treated with ubiquitin beads compared to the control samples (S+B: 52.9±11.9% vs. S-B: 44.5±16.4%). Treated samples did not show an increase of spermatozoa with intact acrosome (S+B: 81.3±13% vs. S-B: 76.5±15.1%), whereas a significant decrease in the total number of spermatozoa (1 x 106sp) was observed after the treatment compared to the control (S+B: 6.7±5.2 vs S-B: 12±7.5; p=0.002). Western Blot analysis using anti-ubiquitin antibodies clearly showed that the same pattern of protein ubiquitination was present before and after treatment with the beads. Actin and prohibitin, and not tubulin, were target of ubiquitin in cat semen and the extent of this post-translational modification was comparable in samples treated with beads and in the control.
CONCLUSIONS. The present data suggest that sperm ubiquitination, morphology and acrosomal integrity of feline epididymal spermatozoa are not related. Sperm anomalies at subcellular level, not revealed by the analyses performed in the present study, should be further investigated before stating that the use of ubiquitin is not a reliable biomarker of semen quality in the domestic cat.
REFERENCES.
(1) Nandi D, Tahiliani P, Kumar A, Chandu D. The ubiquitin-proteasome system. J Biosci 2006;31:137-155.
(2) Baska KM, Manandhar G, Feng D, Agca Y, Tengowski MW, Sutovsky M, Yi YJ, Sutovsky P. Mechanism of extracellular ubiquitination in the mammalian epididymis. J Cell Physio 2008;215:684-696.
(3) Sutovsky P, Hauser R, Sutovsky M. Increased levels of sperm ubiquitin correlate with semen quality in men from an andrology laboratory clinic population. Hum Reprod 2004;19(3):628-38.
(4) Muratori M, Marchiani S, Forti G, Baldi E. Sperm ubiquitination positively correlates to normal morphology in human semen. Hum Reprod 2005;20:1035-1043.
(5) Mota PC, Ramalho-Santos J. Comparison between different markers for sperm quality in the cat: Diff-Quik as a simple optical technique to assess changes in the DNA of feline epididymal sperm. Theriogenology 2006;65:1360–1375.
(6) Sutovsky P, Thompson W. Ubiquitination of prohibitin in mammalian sperm mitochondria: possible roles in the regulation of mitochondrial inheritance and sperm quality control. Biol Reprod 2003;69:254-260
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa
No full textSperm DNA fragmentation is an important parameter to assess sperm quality and can be a
putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle
differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and
require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm
chromatin dispersion test and developed for spermatozoa of different species, but not for
cat spermatozoa, became commercially available. The first aim of the present study was to
verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for
the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose,
DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxy-nucleotidyl transferase–mediated nick-end labeling (TUNEL) were compared. The second
aim was to investigate whether a correlation between DNA status, sperm head
morphology, and morphometry assessed by computer-assisted semen analysis exists in cat
epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable
evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be
independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to
the comp letion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies
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