Biomedical subjects

Pilar Coy

Publications and source records attributed to Pilar Coy.

9 recordsLinked to original sources

PubMed2007

Role of sialic acid in bovine sperm-zona pellucida binding.

Sperm binding activity has been detected in zona pellucida (ZP) glycoproteins and it is generally accepted that this activity resides in the carbohydrate moieties. In the present study we aim to identify some of the specific carbohydrate molecules involved in the bovine sperm-ZP interaction. We performed sperm binding competition assays, in vitro fecundation (IVF) in combination with different lectins, antibodies and neuraminidase digestion, and chemical and cytochemical analysis of the bovine ZP. Both MAA lectin recognising alpha-2,3-linked sialic acid and neuraminidase from Salmonella typhimurium with catalytic activity for alpha-2,3-linked sialic acid, demonstrated a high inhibitory effect on the sperm-ZP binding and oocyte penetration. These results suggest that bovine sperm-ZP binding is mediated by alpha-2,3-linked sialic acid. Experiments with trisaccharides (sialyllactose, 3'-sialyllactosamine and 6'-sialyllactosamine) and glycoproteins (fetuin and asialofetuin) corroborated this and suggest that at least the sequence Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc is involved in the sperm-ZP interaction. Moreover, these results indicate the presence of a sperm plasma membrane specific protein for the sialic acid. Chemical analysis revealed that bovine ZP glycoproteins contain mainly Neu5Ac (84.5%) and Neu5GC (15.5%). These two types of sialic acid residues are probably linked to Galbeta1,4GlcNAc and GalNAc by alpha-2,3- and alpha-2,6-linkages, respectively, as demonstrated by lectin cytochemical analysis. The use of a neuraminidase inhibitor resulted in an increased number of spermatozoa bound to the ZP and penetrating the oocyte. From this last result we hypothesize that a neuraminidase from cortical granules would probably participate in the block to polyspermy by removing sialic acid from the ZP.

Animals↗

PubMed2006

First steps in the development of a functional assay for human sperm using pig oocytes.

The use of mammalian oocytes to assess human sperm functionality could be a helpful tool with potential applications in clinical and research programs. In an attempt to develop the pig model, the aim of the present work was to study the interaction between human spermatozoa and pig oocytes at the zona pellucida (ZP), the oolemma, and the ooplasm levels. In vitro matured pig oocytes and human spermatozoa from fertile and low-fertility donors were employed. The induction of the acrosome reaction by the ZP, the ability of the sperm to penetrate the oocyte after coincubation, and the male pronuclear formation after ICSI were evaluated. Human spermatozoa can bind to pig ZP and undergo the acrosome reaction (15% to 58%, depending on the individual); they are not able to fuse with the oolemma but they can decondense and form a male pronucleus (40%-100%) when injected into pig oocytes. In conclusion, this study shows that pig oocytes can be a useful model to assess human sperm functionality.

Acrosome Reaction↗

PubMed2006

Analysis of different factors influencing the intracytoplasmic sperm injection (ICSI) yield in pigs.

Intracytoplasmic sperm injection (ICSI) in pigs is a technique with potential application in diverse fields of animal production and biomedicine. Even though there are some cases of live offspring resulting from this technique, its yield is still quite low compared to other species. The aim of this study was to evaluate different factors affecting the ICSI performance. This was done by studying (1) the sequence of culture media for the oocytes after injection; (2) modifications in the in vitro maturation system (IVM) through meiotic inhibitors such as roscovitine, and changes in the IVM time; (3) oocyte activation through injection of inositol triphosphate (InsP(3)) together with the sperm. In vitro matured oocytes were employed. All the ICSI experiments were performed with fresh ejaculated semen. Results showed that porcine ICSI zygotes give an improved proportion of two-cell embryos using the sequence IVF medium-embryo culture medium (NCSU-23) rather than transferring directly to NCSU-23. Pronuclear formation ability was not affected by prematuration, but a faster embryo development was observed in roscovitine treated oocytes. In relation to IVM times, oocytes matured for 36 h can achieve better fertilization percentages than oocytes matured for 44 h. These results were independent of the roscovitine treatment. Finally, no influence on embryo development was observed until the blastocyst stage with the use of the InsP(3) as an exogenous activating factor.

Animals↗

PubMed2005

Influence of sperm pretreatment on the efficiency of intracytoplasmic sperm injection in pigs.

The purpose of this study was to determine the influence of sperm pretreatment on the efficiency of intracytoplasmic sperm injection (ICSI) in pigs. This was done by examining the effect of 1) the conservation method (fresh vs frozen); 2) the sperm treatment preinjection (resuspension in Dulbecco phosphate-buffered saline (DPBS) vs selection by a Percoll gradient); and 3) the acrosomal and live or dead status of the spermatozoa (by incubation with or without calcium ionophore, 1 muM and 5 muM). In vitro matured porcine oocytes were injected with treated spermatozoa according to each experiment. All the experiments were done with non-artificially activated oocytes. The percentages of activation and cleavage were higher (68% vs 43% and 63% vs 43%, respectively, P < .05) in oocytes injected with fresh vs frozen spermatozoa. The DPBS treatment allowed higher cleavage proportions than the Percoll treatment (P < .05). Moreover, a boar effect was observed in the percentage of developing blastocysts. None of the studied parameters was affected by the acrosomal or the live or dead status of the spermatozoa injected. In conclusion, the use of fresh semen is recommended for porcine ICSI, as well as careful selection of the boar; Percoll treatment is only recommended for poor-quality samples or for removing toxic agents, and no exogenous form of activation or induction of the acrosome reaction is necessary for porcine oocytes to develop a male pronucleus and cleave up to the 2-cell stage after ICSI, although experimental conditions to reach the blastocyst stage need to be investigated further.

Animals↗

PubMed2005

Maintenance of meiotic arrest in bovine oocytes using the S-enantiomer of roscovitine: effects on maturation, fertilization and subsequent embryo development in vitro.

The overall objective was to evaluate the effectiveness of the S-enantiomer of roscovitine (inhibitor of p34cdc2/cyclin B kinase) to maintain bovine cumulus-oocyte complexes at the germinal vesicle (GV) stage for extended times after removal from antral follicles without compromising subsequent maturation, fertilization and embryo development. Oocytes were cultured in 0, 12.5, 25 or 50 micromol/l S-roscovitine for 24 h. Hoechst staining showed that 50 micromol/l S-roscovitine maintained >90% of oocytes at the GV stage and inhibited gonadotropin-induced cumulus expansion. Fewer oocytes underwent nuclear maturation after in vitro maturation (Hoechst staining) when cultured in 50 micromol/l S-roscovitine for 66 versus 21 or 42 h. Zona pellucida (ZP) hardening (pronase resistance), cortical granule types (lens culinaris agglutinin-fluorescein isothiocyanate), nuclear maturation and fertilization with frozen-thawed spermatozoa (Hoechst staining) were assessed after culture of oocytes in 50 micromol/l S-roscovitine for 0, 24 or 48 h. Neither ZP hardening, nor nuclear maturation nor fertilization were altered by roscovitine culture for 48 h. A higher proportion of oocytes had a type III cortical granule pattern (premature translocation to the oolemma) after roscovitine culture for 48 h. However, embryo development was not compromised as cleavage, development to 8-16 cell and blastocyst stages were at least comparable in control and roscovitine-treated oocytes. In conclusion, the studies have shown that S-roscovitine reversibly maintained bovine oocytes at the GV stage for 48 h. However, maintenance of oocytes in static culture for 48 h was not sufficient to improve development above non-treated controls.

Animals↗

PubMed2005

Birth of piglets after transferring of in vitro-produced embryos pre-matured with R-roscovitine.

The objectives of this study were to evaluate: (1) the nuclear maturation, (2) the intracellular glutathione (GSH) content, (3) the normality of fertilization and (4) full development after transplantation of embryos derived from porcine oocytes pre-cultured with 50 micromol/l roscovitine (an inhibitor of p34cdc2/cyclin B kinase) for 22 h. After treatment with roscovitine, the nuclear configuration of oocytes (Hoechst staining) was comparable with those examined just after collection: the majority of oocytes were arrested at the germinal vesicle (GV) 1 stage (63.2%). Roscovitine-treated oocytes progressed through meiosis to the metaphase II stage in a conventional step-wise in vitro maturation (IVM) program for 44 h in a proportion similar to control ones (>85.0%). When roscovitine-treated oocytes and non-treated oocytes were matured for 44 h and then co-cultured with fresh spermatozoa for 18 h, no differences were observed in oocyte penetrability, proportion of monospermic penetration and male pronuclear formation (>87%). Roscovitine increased the GSH synthesis in oocytes at 22 h, whereas, after 44 h, roscovitine-treated oocytes had similar amounts of GSH to non-treated oocytes. Finally, surgical transfer of zygotes at 22-24 h post-insemination, derived from roscovitine-treated oocytes, resulted in one pregnancy with 12 piglets born; control non-treated zygotes resulted in one pregnancy and 10 piglets born. The full-term developmental ability of mammalian oocytes pre-cultured with roscovitine prior to IVM is thereby demonstrated. This validation is important before the introduction of roscovitine into routine procedures.

Animals↗

PubMed2004

Susceptibility of bovine germinal vesicle-stage oocytes from antral follicles to direct effects of heat stress in vitro.

Delineation of maternal versus direct effects of heat stress in reducing development at the germinal vesicle (GV) stage is challenging, because oocytes spontaneously resume meiosis after removal from antral follicles. The use of S-roscovitine (inhibitor of p34(cdc2)/cyclin B kinase) to hold bovine oocytes at the GV stage without compromising early embryo development was previously validated in our laboratory. The objective of the present study was to assess the direct effects of an elevated temperature commonly seen in heat-stressed dairy cows on cumulus-oocyte complexes (COCs) held at the GV stage using 50 microM S-roscovitine. During roscovitine culture, GV-stage COCs (antral follicle diameter, 3-8 mm) were cultured at 38.5 or 41 degrees C. Thereafter, oocytes were removed from roscovitine medium and allowed to undergo in vitro maturation, fertilization, and culture. Zona pellucida hardening (solubility to 0.5% pronase), nuclear stage (Hoechst 33342), cortical granule type (lens culinaris agglutinin-fluorescein isothiocyanate [FITC]), and early embryo development were evaluated. Culture of GV-stage COCs at 41 degrees C increased the proportion that had type III cortical granules and reduced the proportion that progressed to metaphase II after in vitro maturation. Effects of 41 degrees C on zona pellucida hardening, fertilization (penetration, sperm per oocyte, pronuclear formation, and monospermic and putative embryos), and cleavage of putative zygotes were not noted. However, culture of GV-stage COCs at 41 degrees C for 6 h decreased the proportion of 8- to 16-cell embryos, whereas 41 degrees C for 12 h reduced blastocyst development. In summary, antral follicle COCs are susceptible to direct effects of elevated body temperature, which may account in part for reduced fertility in heat-stressed cows.

Animals↗

PubMed2004

Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders.

Although glutathione content in boar spermatozoa has been previously reported, the effect of reduced glutathione (GSH) on semen parameters and the fertilizing ability of boar spermatozoa after cryopreservation has never been evaluated. In this study, GSH content was determined in ejaculated boar spermatozoa before and after cryopreservation. Semen samples were centrifuged and GSH content in the resulting pellet monitored spectrophotometrically. The fertilizing ability of frozen-thawed boar sperm was also tested in vitro by incubating sperm with in vitro matured oocytes obtained from gilts. GSH content in fresh semen was 3.84 +/- 0.21 nM GSH/10(8) sperm. Following semen cryopreservation, there was a 32% decrease in GSH content (P < 0.0001). There were significant differences in sperm GSH content between different boars and after various preservation protocols (P = 0.0102 ). The effect of addition of GSH to the freezing and thawing extenders was also evaluated. Addition of 5 mM GSH to the freezing extender did not have a significant effect on standard semen parameters or sperm fertilizing ability after thawing. In contrast, when GSH was added to the thawing extender, a dose-dependent tendency to increase in sperm fertilizing ability was observed, although no differences were observed in standard semen parameters. In summary, (i) there was a loss in GSH content after cryopreservation of boar semen; (ii) addition of GSH to the freezing extender did not result in any improvement in either standard semen parameters or sperm fertilizing ability; and (iii) addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Nevertheless, future studies must conclude if this is the case for all boars. Furthermore, since addition of GSH to the thawing extender did not result in an improvement in standard semen parameters, this suggests that during the thawing process, GSH prevents damage of a sperm property that is critical in the fertilization process but that is not measured in the routine semen analysis.

Animals↗

PubMed2002

In vitro production of pig embryos: a point of view.

Porcine embryos have become raw materials for different programmes of reproductive biotechnology and the in vitro production of embryos has some advantages over in vivo production in gene transfer programmes and for xenotransplantation. Despite this promising future, several problems limit the success of the in vitro production (IVP) of viable porcine embryos. Porcine IVP has not been fully developed because of several problems associated with different techniques, such as incomplete final maturation status after in vitro maturation, a high incidence of polyspermy after in vitro fertilization and a low development rate and poor quality of blastocysts at the end of culture. The results could be improved with studies comparing in vivo and in vitro conditions, standardization of techniques for sperm processing, testing new additives in the culture media and developing intracytoplasmic sperm injection procedures. The first objective of the present article is to summarize the main studies published on the subject. Second, we provide a guide for researchers starting work on the IVP of pig embryos, making special mention of first papers and the most recent achievements for each of the different techniques. Third, we provide suggestions for future experiments designed to improve the results of each technique.

Animals↗