Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity

Objective: To investigate effects of cryopreservation on sperm motility and DNA integrity. Design: Pre-cryopreservation and post-cryopreservation analysis of motility and DNA integrity of semen and prepared sperm samples. Setting: A hospital andrology laboratory. Patient(s): Forty men attending the Regional Fertility Centre, Belfast, Northern Ireland. Intervention(s): Each sample was divided, and an aliquot was frozen unprepared. Remaining aliquots were prepared by Percoll density centrifugation (95.0:47.5) or direct swim-up procedure and divided into aliquots to allow direct comparison of fresh and frozen semen and prepared sperm (frozen with or without the addition of seminal plasma) from the same ejaculate. Samples were frozen by static-phase vapor cooling and being plunged into liquid nitrogen. Thawing was carried out at room temperature. Main Outcome Measure(s): Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay, and motility was determined using computer-assisted semen analysis. Result(s): Sperm frozen unprepared in seminal fluid appeared more resistant to freezing damage than frozen prepared sperm. Further improvements can be achieved by selecting out the subpopulation of sperm with best motility and DNA integrity and freezing these sperm in seminal plasma, making this the optimal procedure. Conclusion(s): Freezing sperm in seminal plasma improves postthaw motility and DNA integrity.

Assessment of DNA integrity and morphology of ejaculated sperm from fertile and infertile men before and after cryopreservation

Cryopreservation of human spermatozoa is extensively used in artifical insemination and IVF programmes. Despite various advances in cryopreservation methodology, the recovery rate of functional post thaw spermatozoa remains mediocre, with sperm motility being significantly decreased after freezing. The aim of this study was to investigate the effects of cryopreservation on both DNA integrity and morphology of spermatozoa from fertile and infertile men. Semen samples were obtained from 17 fertile men and 40 infertile men. All samples were prepared by discontinuous Percoll density centrifugation ( 95.0:47.5). Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. Aliquots for cryopreservation were mixed with a commercial cryoprotectant and frozen by static phase vapour cooling before plunging into liquid nitrogen. Thawing was carried out slowly at room temperature. Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis ( comet ) assay and sperm morphology analysed using the Tygerberg criteria. DNA of semen and prepared sperm from fertile men was found to be unaffected by cryopreservation. In marked contrast, spermatozoa from infertile men were significantly damaged by freeze- thawing. Cryopreservation had a detrimental effect on morphology of semen and prepared sperm from fertile and infertile men.

A novel method for isolation of human lung T cells from lung resection tissue reveals increased expression of GAPDH and CXCR6

Lung T lymphocytes are important in pulmonary immunity and inflammation. it has been difficult to study these cells due to contamination with other cell types, mainly alveolar macrophages. We have developed a novel method for isolating lung T cells from lung resection tissue, using a combination of approaches. Firstly the lung tissue was finely chopped and filtered through a nylon mesh. Lymphocytic cells were enriched by Percoll density centrifugation and the T cells purified using human CD3 microbeads, resulting in 90.5% +/- 1.9% (n = 11) pure lymphocytes. The T cell yield from the crude cell preparation was 10.8 +/- 2.1% and viability, calculated using propidium iodide (PI) staining and trypan blue, was typically over 95%. The purification process did not affect expression of CD69 or CD103, nor was there a difference in the proportion of CD4 and CD8 cells between the starting population and the purified cells. Microarray analysis and real time RT-PCR revealed upregulation of GAPDH and CXCR6 of the lung T cells as compared to blood-derived T cells. This technique highly enriches lung T cells to allow detailed investigation of the biology of these cells. (C) 2008 Elsevier B.V. All rights reserved.

Immunochemical characterization of a polysaccharide antigen of Bacteroides fragilis with an IgM monoclonal antibody

An IgM mouse monoclonal antibody (McAb) Bf4 was produced to a surface polysaccharide of Bacteroides fragilis NCTC 9343. Immunoblotting showed that McAb Bf4 reacted strongly with a high molecular mass structure which was sensitive to oxidation with periodate but resisted protease treatment. An inhibition enzyme-linked immunosorbent assay (ELISA) indicated that McAb Bf4 did not cross react with the sixteen Bacteroides species and strains tested. Cells of B. fragilis NCTC 9343 recovered from the various interfaces of a Percoll discontinuous density gradient were tested in the inhibition ELISA. Bacteria from the 0-20%, 20-40% and 40-60% interfaces inhibited the ELISA; however, cells from the 60-80% interface did not. Electron microscopy with immunogold labelling showed that McAb Bf4 did not react with the extracellular fibrous network on bacteria recovered from the 0-20% interface, or the extracellular electron dense layer on cells from the 60-80% interface; however, it was associated with a surface structure on cells from the 20-40% interface. Growth in vivo did not enrich for bacteria with this structure.

A comparison of the haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles

The haemagglutinating and enzymic activities of the obligately anaerobic pathogenic bacterium Bacteroides fragilis were examined. Outer membrane vesicles are released from the surface of B. fragilis. They can be detected by electron microscopy in ultrathin sections and bacterial suspensions after negative staining. Electron microscopy and immunogold labelling with a MAb specific for surface polysaccharide of B. fragilis confirmed that the vesicles carried outer membrane associated epitopes. The haemagglutinating activity of whole cells from populations of B. fragilis strains NCTC9343, BE3 and LS66 enriched by Percoll density gradient centrifugation for a large capsule (LC), electron dense layer (EDL); non-capsulate by light microscopy) and outer membrane vesicles (OMV) which had been purified by centrifugation from EDL-enriched populations were compared using human and horse erythrocytes. The enzymic activity of OMV, LC- and EDL-enriched populations, as detected by the API ZYM kit, was compared for strains NCTC 9343 and BE3. Purified OMV from the strains examined exhibited both haemagglutinating and enzymatic activity. Haemagglutination by the EDL-enriched population was sensitive to treatment with sodium periodate. The LC-enriched population haemagglutinated only after ultrasonic removal of the capsule. This indicates that the LC masks a haemagglutinin. The results suggest a potential role for OMV in the virulence of B. fragilis.

Thérapie par les cellules souches mésenchymateuses dans la guérison tendineuse chez le cheval

Les tendinites sont des lésions communes chez le cheval athlète, ayant un impact financier et sportif considérable. Les cellules souches mésenchymateuses (CSMs) de moelle osseuse (MO) sont empiriquement utilisées en clinique pour améliorer la guérison des affections myoarthrosquelettiques. Cependant, il est nécessaire de standardiser les protocoles d’isolement des CSMs équines et d’analyser leurs effets sur la guérison tendineuse pour ajuster leur dose. Les objectifs de cette étude étaient de comparer 3 méthodes d’isolement des CSMs équines et d’établir un modèle de guérison tendineuse minimal invasif pour analyser l’effet des CSMs sur cette guérison. Des CSMs de MO du sternum de juments étaient isolées par 3 protocoles couramment utilisés (adhérence au pétri (Classique) et 2 méthodes par gradient de densité (Percoll et Ficoll)). La viabilité des cellules après isolement, le rendement d’isolement, le nombre de CSMs obtenues après 14 jours de culture et leurs caractéristiques fonctionnelles (renouvellement et différentiation) étaient comparés entre les 3 protocoles. Les résultats suggéraient que le Percoll était le meilleur protocole en termes de rendement et de capacité de renouvellement des cellules. La différence n’était pas significative pour leur viabilité et leur capacité de différentiation. Un modèle de guérison tendineuse, consistant en une ténectomie du tendon extenseur latéral du doigt fut ensuite développé. Cependant, la grande variabilité interindividuelle de qualité de guérison dans le groupe pilote implique une ré-évaluation du modèle. Des études futures, avec des CSMs isolées par le Percoll dans de nouveaux modèles de guérison tendineuse devraient permettre de déterminer la dose adéquate de CSMs.

Fecundação in vitro de ovócitos bovinos com sêmen submetido a diferentes diluidores

O objetivo deste trabalho foi avaliar a influência do diluidor do sêmen no desenvolvimento in vitro de ovócitos bovinos após a maturação e fecundação in vitro. Ejaculado de um reprodutor foi fracionado e submetido a três diluidores: Lactose/gema de ovo (LG), Citrato/gema de ovo (CG) e Tris/gema de ovo (TG). Amostras deste material foram envasadas, congeladas e estocadas em N² e, posteriormente, descongeladas; a fração móvel foi separada por gradiente descontínuo de Percoll. A concentração espermática foi ajustada para 10 x 10(6)/mL e a capacitação espermática, induzida com 10 µg/mL de heparina. Após 24 horas de cultura para maturação in vitro, os ovócitos, aspirados de folículos ovarianos, foram inseminados com sêmen diluído em meio TALP e, após 48 horas de cultura, os zigotos foram transferidos para gotas de meio TCM 199, com 5% de soro fetal bovino, 5% de soro de vaca em estro e suspensão de células epiteliais do oviduto bovino, cobertas com óleo de silicone, e mantidos em cultura por nove dias. Todas as culturas foram realizadas a 38,5ºC em atmosfera com 5% de CO2. Os dados foram analisados pelo teste do qui-quadrado e houve diferença com relação à taxa de clivagem (TC), sendo as médias de 66,0; 69,3; e 54,4% para LG, CG e TG, respectivamente. Não houve diferença entre tratamentos com relação às taxas de mórulas/blastocistos ou de eclosão. O diluidor do sêmen não teve efeito sobre o desenvolvimento in vitro de embriões bovinos, embora a TC tenha sido afetada.

Comparative validation using quantitative real-time PCR (qPCR) and conventional PCR of bovine semen centrifuged in continuous density gradient

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Possible role of bovine trophoblast giant cells in transplacental transmission of Neospora caninum in cattle

Neospora caninum is an aplicomplexan parasite that has brought several concerns to cattle raisers worldwide due to its relationship to fetal loss. However, the mechanism of the parasite's transplacental infection and induced abortions are not completely understood. Bovine trophoblastic binucleated cells (BNC) play a major role in the maternal-fetal interactions, migrating during the entire pregnancy from chorionic connections to uterine epithelium. This study aimed to investigate the possible role of BNC as phagocytic cells and its participation in the bovine transplacental infection of N. caninum. BNC was isolated by discontinuous Percoll gradient, and characterized by Hoeschst 33342 nucleus-specific staining. Isolated BNC were cultured in DMEM supplemented with 10% bovine fetal serum, and infected with 10(4) tachyzoites of N. caninum NC-1 strain. Parasite invasion was visualized by indirect immunofluorescence and Giemsa technique. Multiplication of parasites took place in 2-3 day cycles. Healthy cows' placenta and normal and infected cultured BNC was immunostained with monoclonal antibodies against CD-163, MAC-387 and NOS, demonstrating their phagocyte capacity. Thus, BNC was characterized as cells with macrophagic activity, which may host N. caninum in vitro. Therefore, we may conclude that BNC could potentially participate in the transplacental infection of bovine neosporosis.

Effects of scrotal insulation in Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability

The objectives of this study were to assess the effects of induced testicular degeneration in Bos taurus indicus (Nellore) bulls on changes in seminal characteristics and fertilizing ability of sperm. Four Nellore bulls (30-36-month-old, 500-550 kg) with good seminal quality (> 80% motile and morphologically normal sperm) had serotal insulation applied for 5 d. Semen was collected by electroejaculation and cryopreserved at a the pre-insulation moment, and 7, 14, and 21 d after insulation was removed. Gross motility, vigor of sperm movement (1-5), acrosome integrity, sperm morphology (phase-contrast microscopy), nuclear vacuoles and abnormal chromatin (Feulgen-stain) were determined after sperm preparations for in vitro fertilization (IVF). Prior to IVF, sperm were separated using a Percoll gradient (45% and 90%). Normal sperm decreased (P < 0.05) 14 and 21 d after insulation was removed. on 14 and 21 d, the incidence of head defects (9.7 +/- 0.6 and 17.0 +/- 0.8, respectively; mean +/- S.E.M) was higher (P < 0.05) in agreement with the incidence of nuclear vauoles (14.0 +/- 5.0 and 12.3 +/- 2.3) and abnormal chromatin (24.4 +/- 7.2 and 30.8 +/- 2.8). Although the frequency of cleaved oocytes decreased only on 21 d (P < 0.05), blastocyst rates were lower (P < 0.05) than pre-insulation on 14 and 21 d. In regression analyses, only nuclear vacuoles, head defects and intact acrosome accounted for differences in cleavage (R(2) = 0.38, 0.48, and 0.30, respectively) and blastocyst rates (R(2) = 0.35, 0.37, and 0.44). Abnormal chromatin was associated only with blastocyst rates (R(2) = 0.35). In conclusion, blastocyst rate was more sensitive than cleavage rate and the assessment of nuclear integrity is recommended to predict the fertilizing ability of bull sperm. (c) 2008 Elsevier B.V. All rights reserved.

Penetration of zona-free hamster, bovine and equine oocytes by stallion and bull spermatozoa pretreated with equine follicular fluid, dilauroylphosphatidylcholine or calcium ionophore A23187

Experiments evaluated the ability of follicular fluid (FF), dilauroylphosphatidylcholine (PC12) and the calcium ionophore A23187 (A23187) to induce capacitation in stallion and bull spermatozoa, determined by the ability of the spermatozoa to penetrate zona-free hamster, bovine and equine oocytes. Spermatozoa suspensions were incubated at 37 degreesC in one of the following treatments: 1) a modified Tyrode's medium (BGM3) alone, 2) BGM3 + FF; 3) BGM3 + PC12; 4) BGM3 + FF + PC12; 5) BGM3 + A23187; and 6) BGM3 + FF + A23187. Treated spermatozoa were incubated with zona-free hamster, bovine and equine oocytes for 3 h, after which oocytes were stained to assess spermatozoa penetration. The number of hamster oocytes penetrated by spermatozoa incubated in BGM3 alone (1/30) or in presence of FF (2/31) was significantly lower (P < 0.05) than by spermatozoa treated with PC12 or A23187 (16/30 and 17/30, respectively). Processing stallion spermatozoa either by a swim-up procedure or by centrifugation through a Percoll gradient increased the percentages of motile spermatozoa in the final sample, and spermatozoa collected by both processes penetrated similar numbers of zona-free hamster oocytes (P > 0.05). Although treating spermatozoa with PC12 or A23187 enabled both stallion and bull spermatozoa to penetrate oocytes, higher numbers of bovine oocytes were penetrated by bull spermatozoa (25/30) than by stallion spermatozoa (4/30) regardless of spermatozoal treatment. However, the number of zona-free hamster and equine oocytes penetrated by bull spermatozoa (25/30 and 12/18 respectively) and stallion spermatozoa (17/30 and 15/21 respectively) were similar (P > 0.05). We conclude that both PC12 and A23187 capacitate stallion and bull spermatozoa sufficiently to permit the acrosome reaction to occur, enabling spermatozoa to penetrate homologous and heterologous zona-free oocytes. (C) 2001 by Elsevier B.V.

Tritrichomonas fetus extracellular products decrease progressive motility of bull sperm

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Coat protein and RNAs of Cole latent virus are not present within chloroplasts of Chenopodium quinoa-infected cells

O vírus latente da couve (Cole latent virus, CoLV), gênero Carlavirus, foi estudado, por microscopia eletrônica de transmissão e técnicas bioquímicas, em relação à ultra-estrutura das células infetadas de Chenopodium quinoa, e de sua associação com os cloroplastos. O CoLV foi observado como partículas dispersas pelo citoplasma entremeadas com vesículas membranosas e ribossomos e/ou como densas massas de partículas. Estes partículas reagiram por imunomarcação com anti-soro policlonal para o CoLV. Morfologicamente, cloroplastos, mitocôndrias e núcleos mostraram-se inalterados e partículas virais não foram encontradas dentro dessas organelas. Entretanto, agregados de partículas virais foram freqüentemente vistos em associação com a membrana externa dos cloroplastos e ocasionalmente com peroxissomos. Cloroplastos foram purificados em gradiente de Percoll e as proteínas e os RNA foram extraídos e analisados, respectivamente, por Western blot e Northern blot. Proteína capsidial e RNA associados ao CoLV não foram detectados nessa organela. Os resultados aqui obtidos indicam que a associação CoLV/cloroplastos, observada nos estudos de microscopia eletrônica, é possivelmente um evento casual dentro da célula hospedeira e que o vírus não se multiplica dentro dessa organela.

Efeito da centrifugação sobre a qualidade do sêmen canino

Avaliou-se o efeito da centrifugação sobre a viabilidade do sêmen canino e compararam-se três meios de diluição pré-centrifugação. Utilizaram-se 10 ejaculados completos de 10 cães que, após a avaliação inicial, foram divididos em quatro porções (grupos). Uma das amostras, não centrifugada, formou o grupo-controle; as outras foram diluídas em três diferentes meios e centrifugadas a 800 x g por 15 minutos, formando os grupos: CPSA - constituído por sêmen centrifugado em plasma seminal autólogo; CLG - sêmen centrifugado em meio à base de leite desnatado e glicose (LG); e CPer- sêmen centrifugado em gradientes de Percoll (45% e 90%). Após a centrifugação e a eliminação do sobrenadante, procedeu-se à ressuspensão de todas as porções do ejaculado em LG e à imediata avaliação quanto à motilidade, vigor, aglutinação espermática e integridade das membranas espermáticas. Todas as suspensões foram, então, incubadas a 37ºC por 30 minutos e reavaliadas. O processo de centrifugação não causou danos aos espermatozoides e a centrifugação em meio LG melhorou a viabilidade espermática.

Human eosinophil adhesion and degranulation stimulated with eotaxin and RANTES in vitro: Lack of interaction with nitric oxide

Background: Airway eosinophilia is considered a central event in the pathogenesis of asthma. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction. Previous studies have suggested an interaction between nitric oxide (NO) and chemokines in modulating eosinophil functions, but this is still conflicting. In the present study, we have carried out functional assays (adhesion and degranulation) and flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) to evaluate the interactions between NO and CC-chemokines (eotaxin and RANTES) in human eosinophils. Methods: Eosinophils were purified using a percoll gradient followed byimmunomagnetic cell separator. Cell adhesion and degranulation were evaluated by measuring eosinophil peroxidase (EPO) activity, whereas expression of Mac-1 and VLA-4 was detected using flow cytometry. Results: At 4 h incubation, both eotaxin (100 ng/ml) and RANTES (1000 ng/ml) increased by 133% and 131% eosinophil adhesion, respectively. L-NAME alone (but not D-NAME) also increased the eosinophil adhesion, but the co-incubation of L-NAME with eotaxin or RANTES did not further affect the increased adhesion seen with chemokines alone. In addition, L-NAME alone (but not D-NAME) caused a significant cell degranulation, but it did not affect the CC-chemokine-induced cell degranulation. Incubation of eosinophils with eotaxin or RANTES, in absence or presence of L-NAME, did not affect the expression of VLA-4 and Mac-1 on eosinophil surface. Eotaxin and RANTES (100 ng/ml each) also failed to elevate the cyclic GMP levels above baseline in human eosinophils. Conclusion: Eotaxin and RANTES increase the eosinophil adhesion to fibronectin-coated plates and promote cell degranulation by NO-independent mechanisms. The failure of CC-chemokines to affect VLA-4 and Mac-1 expression suggests that changes in integrin function (avidity or affinity) are rather involved in the enhanced adhesion. © 2008 Lintomen et al; licensee BioMed Central Ltd.