Effect of different freezing velocities on the quality and fertilising ability of cryopreserved rabbit spermatozoa.

The freezing step of the cryopreservation protocol negatively influences the quality and fertilising ability of rabbit spermatozoa. This study determines the effect of different rates of freezing on the quality and fertilising ability of rabbit spermatozoa cryopreserved with dimethylsulfoxide (DMSO) (1.75M) and sucrose (0.05M). Ejaculates from meat rabbit line males (n=12) were pooled and each pool (n=7) was split into four aliquots. One group of straws (control, C) was frozen in static liquid nitrogen vapour (5cm above the liquid nitrogen, 10min) and the other groups were frozen at different freezing rates (°Cmin(-1)) from -6°C to -100°C using a programmable freezer: slow (-15°Cmin(-1), S), medium (-40°Cmin(-1), M) or fast (-60°Cmin(-1), F). After thawing (50°C, 12s), the quality was highest (P<0.05) in C and M samples and lowest in S and F samples. F samples presented the lowest litter sizes (P≤0.05) and fertility whilst M samples exhibited the highest values. In conclusion, the freezing rate affects both the quality and the fertilising ability of frozen-thawed rabbit spermatozoa, with both slow (-15°Cmin(-1)) and fast (-60°Cmin(-1)) freezing rates being detrimental for the quality and fertilising ability.

Reducing the time rabbit sperm are held at 5 °C negatively affects their fertilizing ability after cryopreservation.

Cooling sperm to and equilibrating the sperm at 5 °C require the most time in any sperm cryopreservation protocol. Reducing the time required for these phases would simplify sperm freezing protocols and allow greater number of ejaculates to be processed and frozen in a given time. This study determined how holding rabbit sperm at 5 °C for different lengths of time (0, 10, 15, 20, 30, or 45 minutes) affected the quality of rabbit sperm, measured by in vitro assays, and if reducing the cooling time to only 10 minutes affected the fertilizing ability of the sperm. Reducing the time sperm were held at 5 °C to 10 minutes did not affect the in vitro quality of the sperm (percent motile and with intact plasma membranes), although eliminating the cooling phase completely (directly freezing the sperm from room temperature) decreased in vitro assessed sperm quality (P<0.01). However, reducing the time sperm were held at 5 °C, from 45 to 10 minutes, negatively affected the fertilizing ability of sperm in vivo (P<0.05). In conclusion, completely eliminating cooling rabbit sperm to 5 °C before freezing is detrimental for rabbit sperm cryosurvival, and although shortening the time sperm are held at 5 °C to 10 minutes does not reduce in vitro sperm quality, it does reduce the fertility of rabbit sperm. Therefore, the length of time rabbit sperm equilibrate at 5 °C is crucial to the fertilizing ability of rabbit sperm and must be longer than 10 minutes. Currently, it is not known if holding rabbit sperm at 5 °C for less than 45 minutes will affect sperm fertilizing ability.

Development of methods for cryopreservation of rooster sperm from the endangered breed "Gallina Valenciana de Chulilla" using low glycerol concentrations.

Glycerol (11%; v:v) is the cryoprotectant most often used for the cryopreservation of rooster sperm. However, chicken breeds differ in the resistance of their sperm to the cryopreservation process and endangered or local breeds usually present low fertilizing ability when conventional sperm cryopreservation protocols are used. The objective of this study was to optimize the protocol for the cryopreservation of the sperm from the endangered breed "Gallina Valenciana de Chulilla". For this purpose, 10 pools of semen from 43 roosters of this breed were cryopreserved using 8%, 7%, 6%, or 4% glycerol, and the sperm quality was determined immediately after thawing and in the insemination doses. Lohmann Brown Classic laying hens (n = 40) were used for the insemination trials. The sperm quality after cryopreservation progressively decreased as the glycerol concentration was reduced (P < 0.01); samples frozen using 4% glycerol exhibited the lowest quality (38% total motile sperm and 49% live sperm), and samples frozen using 8% glycerol exhibited the highest quality (67% total motile sperm and 66% live sperm). These differences were also observed after the glycerol was removed (P < 0.01). However, the sperm fertilizing ability was similar for all the treatments (23%-30% fertilized eggs), and increased as the glycerol concentration decreased. In conclusion, semen from roosters frozen using 4% glycerol exhibited lower sperm quality but similar fertilizing ability compared with samples processed using higher glycerol concentrations. These results may provide useful information for developing cryopreservation protocols for other breeds.

Effect of cholesterol-loaded cyclodextrins on bull and goat sperm processed with fast or slow cryopreservation protocols.

Cholesterol-loaded cyclodextrins (CLC) added to the sperm before cryopreservation enhance sperm quality after freeze-thawing in several cold shock-sensitive species, including cattle and goats. However, all studies conducted to date have used conventional protocols, in which sperm are cooled slowly to 5°C before freezing. As cholesterol plays a significant role in sperm cold shock resistance, it is possible that CLC-treated sperm can withstand cooling damage when the sperm are not cooled slowly to 5°C before freezing. In this study, we determined whether CLC-treated goat (1 mg CLC/120×10(6) sperm) and bull (2 mg CLC/120×10(6) sperm) sperm quality, after thawing, was different for sperm frozen using conventional protocols (including a slow cooling phase to 5ºC) and protocols in which the sperm were frozen from room temperature, without cooling the sperm slowly to 5°C before freezing. CLC-treated sperm exhibited higher percentages of plasma membrane-intact sperm than control sperm when cryopreserved using conventional protocols. In addition, CLC treatment enhanced both sperm motility and plasma membrane integrity when sperm were frozen directly from room temperature. However, this treatment did not fully prevent the damage of the sperm after cooling rapidly and subsequent freezing, as the sperm quality was lower than that presented by the samples frozen using the conventional protocol. The results are promising, but studies to optimize the protocols for freezing sperm directly from room temperature need to be conducted, as well as studies to determine how cryopreserving sperm in this manner affects other sperm functions.

Understanding glycoprotein behaviours using Raman and Raman optical activity spectroscopies: characterising the entanglement induced conformational changes in oligosaccharide chains of mucin.

We illustrate the great potential of Raman and ROA spectroscopies for investigating the structure and organisation of glycoproteins and the complex matrices they can form. In combination these spectroscopic techniques are sensitive to changes in conformation revealing details of secondary and tertiary structures, probing hydrogen bonding interactions, as well as resolving side chain orientation and the absolute configuration of chiral substructures. To demonstrate this potential we have characterised the structural changes in a complex glycoprotein, mucin. Spectral changes were observed during the entanglement transition as the mucin concentration was increased. By applying two-dimensional correlation analysis (2DCos) to the ROA and Raman concentration-dependent spectral sets delicate transitions in mucin conformation could also be determined. From ~20-40 mg/ml conformational transitions assigned mainly to the sugar N-acetyl-d-galactosamine (GalNAc), which is the linking saccharide unit to the protein backbone, were monitored. Further changes in local oligosaccharide conformation above 40 mg/ml were also monitored, together with other structural transitions observed in the protein core, particularly ß-structure formation. Consequently, these spectral techniques were shown to monitor the formation of transient entanglements formed by brush-brush interactions between oligosaccharide combs of mucin molecules identifying changes in both carbohydrate and protein moieties. This work clearly shows how these methods can be used to elucidate fresh insights into the complex behaviour of these large complex molecules.

Optimizing conditions for treating goat semen with cholesterol-loaded cyclodextrins prior to freezing to improve cryosurvival.

The fertility of goat sperm is highly variable and new methods for improving sperm cryosurvival are needed. Cholesterol plays important roles in membrane fluidity, cold shock sensitivity and cryodamage, and treating sperm from cold-shock sensitive species with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances sperm cryosurvival. The aim of this study was to develop a CLC-treatment to optimize goat sperm cryopreservation. A total of 45 ejaculates coming from eleven adult Murciano-Granadina bucks were used and three experiments were conducted to determine: (1) the optimal CLC concentration to treat goat sperm; (2) the optimal time to treat the sperm (before or after seminal plasma removal); and (3) optimal freezing diluent (either of two Tris-citrate diluents containing 2% or 20% egg yolk and 4% glycerol or a skim milk diluent with 7% glycerol) to cryopreserve goat sperm. Goat sperm cryosurvival rates were greatest when they were treated with 1mg CLC/120 × 10(6)sperm prior to freezing. The benefit was also greatest if the sperm were treated with CLC after seminal plasma removal. Finally, CLC treatment improved sperm cryosurvival rates for sperm frozen in all three diluents, however, CLC treatment was most effective for sperm frozen in egg-yolk diluents. In conclusion, treating goat sperm, with CLC prior to cryopreservation, improved sperm cryosurvival rates. In addition, CLC treatment was effective for all freezing diluents tested, making this technology practical for the industry using current cryopreservation techniques. Nevertheless, additional studies should be conducted to determine how CLC might affect sperm functionality and fertilizing ability.

Effect of a pre-freezing treatment with cholesterol-loaded cyclodextrins on boar sperm longevity, capacitation dynamics, ability to adhere to porcine oviductal epithelial cells in vitro and DNA fragmentation dynamics.

The aim of this work was to examine how a pre-freezing treatment with cholesterol-loaded cyclodextrins (CLC) affects boar sperm longevity, capacitation dynamics, ability to bind to a porcine telomerase-immortalised oviductal epithelial cell line (TERT-OPEC) in vitro and DNA integrity dynamics after freeze-thawing. Although the samples treated with CLC exhibited lower sperm quality than the control samples (P<0.05) immediately after thawing, these differences disappeared (P>0.05) after long-term incubation (26h at 37 or 16°C). Additionally, the CLC-treated spermatozoa underwent similar capacitation and DNA fragmentation dynamics as the control spermatozoa (P>0.05). However, CLC-treated spermatozoa were better able to bind to TERT-OPEC in vitro (P<0.0001). In conclusion, the pre-freezing treatment of boar spermatozoa with CLC enhanced the ability of the spermatozoa to bind to TERT-OPEC in vitro, which could have an effect on the establishment of the sperm reservoir in the ampullary--isthmic junction in vivo. Additionally, frozen-thawed spermatozoa can be stored at 16°C for at least 6h without a significant observable decline in sperm quality, which could be beneficial for the transport of thawed diluted doses of spermatozoa from the laboratory to the farm.

Response of boar sperm to the treatment with cholesterol-loaded cyclodextrins added prior to cryopreservation.

Cryopreserved boar sperm is not used extensively for artificial insemination, owing to the poor fertility rates of the sperm after freezing and thawing. The sperm membrane is damaged as the cells are cooled from body temperature to 5°C (cold shock), as well as during the freeze-thaw process. Increasing the cholesterol content of boar sperm membranes could help them survive cryopreservation, similar to sperm from other species that are cold shock sensitive. The aim of this study was to determine the optimal cholesterol-loaded cyclodextrin (CLC) concentration to use for boar sperm cryopreservation, and the influence of CLCs on the cryosurvival of sperm from boars classified as good or poor freezers. Treating boar sperm with 1 mg of CLC/120 × 10(6) sperm slightly improved (p < 0.05) the percentage of viable sperm after freezing-thawing. On the other hand, sperm, from both good and poor freezers, responded similarly to CLC treatment. Nevertheless, additional studies will be needed to study the effect of this treatment on other parameters of sperm quality.

Use of cholesterol in sperm cryopreservation: present moment and perspectives to future.

CONTENTS: Sperm cryosurvival rates are not optimal for most species. Therefore, new cryopreservation strategies are needed with the objective of increasing the number of surviving sperm and the quality of those sperm after thawing. Cholesterol plays important roles in many sperm functions, including effects on membrane properties. One of these effects is to stabilize membranes at low temperatures. Thus, species that produce sperm which possess high membrane cholesterol : phospholipid ratios are more resistant to cold shock than sperm with low cholesterol : phospholipid ratios. Therefore, increasing the cholesterol content of sperm membranes may be a strategy that can improve sperm quality after freeze-thawing. In this review, information is presented related to using cyclodextrins pre-loaded with cholesterol for cryopreserving sperm from different species. The topics discussed include both in vitro and in vivo assessments of sperm quality after cryopreservation, as well as how increasing sperm cholesterol content affects other sperm functions.

Investigation of polypeptide conformational transitions with two-dimensional Raman optical activity correlation analysis, applying autocorrelation and moving window approaches.

The study of conformational transitions in polypeptides is not only important for the understanding of folding mechanisms responsible for the self-assembly of proteins but also for the investigation of the misfolding of proteins that can result in diseases including cystic fibrosis, Alzheimer's, and Parkinson's diseases. Our recent studies developing two-dimensional Raman optical activity (ROA) correlation analysis have proven to be successful in the investigation of polypeptide conformational transitions. However, the complexity of the ROA spectra, and the 2D correlation synchronous and asynchronous plots, makes data analysis detailed and complex, requiring great care in interpretation of 2D correlation rules. By utilizing the 2D correlation approaches of autocorrelation and moving windows it has been possible to gain further information from the ROA spectral data sets in a simpler and more consistent way. The most significant spectral intensity changes have been easily identified, facilitating appropriate interpretation of synchronous plots, and transition phases have been identified in the moving window plots, directly relating spectral intensity changes to the perturbation.

Embarazo abdominal complicado del primer trimestre / Complicated abdominal pregnancy of the first trimester

Relatamos un caso de embarazo abdominal que se complicó con hemoperitoneo ocurrido en el primer trimestre. Ambas trompas, ovarios y el útero se encontró intacto. El sitio de implantación era el fondo de saco de Douglas, en la superficie anterior del recto.

Solution structure of native proteins with irregular folds from Raman optical activity.

Raman optical activity (ROA) spectra have been measured for the proteins hen phosvitin, yeast invertase, bovine alpha-casein, soybean Bowman-Birk protease inhibitor, and rabbit Cd(7)-metallothionein, all of which have irregular folds in the native state. The results show that ROA is able to distinguish between two types of disorder. Specifically, invertase, alpha-casein, the Bowman-Birk inhibitor, and metallothionein appear to possess a "static" type of disorder similar to that in disordered states of poly(L-lysine) and poly(L-glutamic acid); whereas phosvitin appears to possess a more "dynamic" type of disorder similar to that in reduced (unfolded) lysozyme and ribonuclease A and also in molten globule protein states. In the delimiting cases, static disorder corresponds to that found in loops and turns within native proteins with well-defined tertiary folds that contain sequences of residues with fixed but nonrepetitive phi,psi angles; and dynamic disorder corresponds to that envisaged for the model random coil in which there is a distribution of Ramachandran phi,psi angles for each amino acid residue, giving rise to an ensemble of interconverting conformers. In both cases there is a propensity for the phi,psi angles to correspond to the alpha, beta and poly(L-proline) II (PPII) regions of the Ramachandran surface, as in native proteins with well-defined tertiary folds. Our results suggest that, with the exception of invertase and metallothionein, an important conformational element present in the polypeptide and protein states supporting the static type of disorder is that of the PPII helix. Long sequences of relatively unconstrained PPII helix, as in alpha-casein, may impart a plastic (rheomorphic) character to the structure.

Amaurosis bilateral de origen cortical en preeclampsia severa / Cortical blindness in severe preeclampsia

Se presenta un caso clínico de amaurosis bilateral cortical en una primigesta con 35 semanas de embarazo asociado a preeclampsia severa. Se demostró radiológicamente la presencia de lesión cortical occipital. Una vez interrumpido el embarazo la paciente recuperó la agudeza visual y es dada de alta en buenas condiciones junto al recién nacido

Prolapso de cúpula vaginal / Prolapse of vaginal vault

El prolapso de cúpula vaginal constituye una complicación tardía de la histerectomía. Para su reparación se han descrito múltiples técnicas, tanto abdominales como vaginales. Se muestra nuestra experiencia en la reparación de este defecto utilizando la colposuspensión al promontorio con malla de Marlex. Se realizó un seguimiento de entre 1 y 2 años y se observó una tasa de curación subjetiva y objetiva de un 100 por ciento con un reinicio de la actividad sexual satisfactoria en 6/8 pacientes, y escasas complicaciones asociadas. Se concluye que esta técnica es una alternativa de elección en pacientes con prolapso de cúpula vaginal

Lupus eritematoso sistémico y embarazo: experiencia clínica en 12 años / Systemic lupus erithematosus and pregnancy: clinical experience in 12 years

En el período de julio de 1988 a agosto de 2000 se atendieron 23 pacientes lúpicas con 24 embarazos en la Maternidad del Barros Luco-Trudeau: la edad fluctuó entre los 18 y 41 años con una mediana de 23 años, 13 (56,5 por ciento) correspondieron a primíparas y 10 (43,5 por ciento) a multíparas. La edad promedio al diagnóstico de lupus fue 21 años. Hubo 8 embarazos con lupus activo en 4 de los cuales el lupus debutó en éste. No hubo actividad lúpica durante el puerperio, presentándose 3 casos en el primer trimestre, 3 en el segundo y 2 en el tercero. Hubo 20 partos de pretérmino (83,3 por ciento) y 7 casos de RCIU (29,1 por ciento). El 58,3 por ciento de los recién nacidos pesaron menos de 2500 g, hubo un 16,6 por ciento de letalidad perinatal (4 casos) y el 75 por ciento de los embarazos se resolvieron en cesárea

Is polyproline II helix the killer conformation? A Raman optical activity study of the amyloidogenic prefibrillar intermediate of human lysozyme.

The amyloidogenic prefibrillar partially denatured intermediate of human lysozyme, prepared by heating the native protein to 57 degrees C at pH 2.0, was studied using Raman optical activity (ROA). A positive band in the room temperature ROA spectrum of the native protein at approximately 1345 cm(-1), assigned to a hydrated form of alpha-helix, is not present in that of the prefibrillar intermediate, where a new strong positive band at approximately 1318 cm(-1) appears instead that is assigned to the poly(l-proline) II (PPII)-helical conformation. A sharp negative band at approximately 1241 cm(-1) in the native protein, assigned to beta-strand, shows little change in the ROA spectrum of the prefibrillar intermediate. The disappearance of a positive ROA band at approximately 1551 cm(-1) assigned to vibrations of tryptophan side-chains indicates that major conformational changes have occurred among the five tryptophan residues present in human lysozyme, four of which are located in the alpha-domain. The various ROA data suggest that a substantial loss of tertiary structure has occurred in the prefibrillar intermediate and that this is located more in the alpha-domain than in the beta-domain. There is no evidence for any increase in beta-structure. The ROA spectrum of hen lysozyme, which does not form amyloid fibrils so readily, remains much more native-like on heating to 57 degrees C at pH 2.0. The thermal behaviour of the alanine-rich alpha-helical peptide AK21 in aqueous solution was found to be similar to that of human lysozyme. Hydrated alpha-helix therefore appears to readily undergo a conformational change to PPII structure on heating, which may be a key step in the conversion of alpha-helix into beta-sheet in the formation of amyloid fibrils in human lysozyme. Since it is extended, flexible, lacks intrachain hydrogen bonds and is fully hydrated in aqueous solution, PPII helix has the appropriate characteristics to be implicated as a critical conformational element in many conformational diseases. Disorder of the PPII type may be a sine qua non for the formation of regular fibrils; whereas the more dynamic disorder of the random coil may lead only to amorphous aggregates.

Raman optical activity characterization of native and molten globule states of equine lysozyme: comparison with hen lysozyme and bovine alpha-lactalbumin.

Vibrational Raman optical activity (ROA) spectra of the calcium-binding lysozyme from equine milk in native and nonnative states are measured and compared with those of the homologous proteins hen egg white lysozyme and bovine alpha-lactalbumin. The ROA spectrum of holo equine lysozyme at pH 4.6 and 22 degrees C closely resembles that of hen lysozyme in regions sensitive to backbone and side chain conformations, indicating similarity of the overall secondary and tertiary structures. However, the intensity of a strong positive ROA band at approximately 1340 cm(-1), which is assigned to a hydrated form of alpha helix, is more similar to that in the ROA spectrum of bovine alpha-lactalbumin than hen lysozyme and may be associated with the greater flexibility and calcium-binding ability of equine lysozyme and bovine alpha-lactalbumin compared with hen lysozyme. In place of a strong sharp positive ROA band at approximately 1300 cm(-1) in hen lysozyme that is assigned to an alpha helix in a more hydrophobic environment, equine lysozyme shows a broader band centered at approximately 1305 cm(-1), which may reflect greater heterogeneity in some alpha-helical sequences. The ROA spectrum of apo equine lysozyme at pH 4.6 and 22 degrees C is almost identical to that of the holo protein, which indicates that loss of calcium has little influence on the backbone and side chain conformations, including the calcium-binding loop. From the similarity of their ROA spectra, the A state at pH 1.9 and both 2 and 22 degrees C and the apo form at pH 4.5 and 48 degrees C, which are partially folded denatured (molten globule or state A) forms of equine lysozyme, have similar structures that the ROA suggests contain much hydrated alpha helix. The A state of equine lysozyme is shown by these results to be more highly ordered than that of bovine alpha-lactalbumin, the ROA spectrum of which has more features characteristic of disordered states. A positive tryptophan ROA band at approximately 1551 cm(-1) in the native holo protein disappears in the A state, which is probably due to the presence of nonnative conformations of the tryptophans associated with a previously identified cluster of hydrophobic residues.

Solution structure and dynamics of biomolecules from Raman optical activity.

Raman optical activity (ROA) measures vibrational optical activity by means of a small difference in the intensity of Raman scattering from chiral molecules in right and left circularly polarized incident laser light. The ROA spectra of a wide range of biomolecules in aqueous solution can now be measured routinely. Because of its sensitivity to the chiral elements of biomolecular structure, ROA provides new information about solution structure and dynamics complementary to that supplied by conventional spectroscopic techniques. This article provides a brief introduction to the theory and practice of ROA spectroscopy followed by a review of recent ROA results on polypeptides, proteins, carbohydrates, nucleic acids and viruses which illustrate how new insight into current problems of structure, folding and function may be obtained from ROA studies.