Biomedical subjects

Raquel Romar

Publications and source records attributed to Raquel Romar.

8 recordsLinked to original sources

PubMed2005

In vitro maturation and fertilization of porcine oocytes after a 48 h culture in roscovitine, an inhibitor of p34cdc2/cyclin B kinase.

Maintaining oocytes at the germinal vesicle (GV) stage in vitro may permit enhanced acquisition of the developmental competence. The objective of the current study was to evaluate the nuclear and cytoplasmic maturation in vitro of porcine oocytes after pretreatment with S-roscovitine (ROS). Cumulus oocyte complexes (COC) were treated with 50 microM ROS for 48 h and then matured for various lengths of time in a conventional step-wise in vitro maturation (IVM) system by using dibutyryl cyclic AMP. The COC that were matured in the same system for 44 h without pretreatment with ROS were used as the control group. At various periods after the start of IVM, oocytes were assessed for the meiotic stages and subjected to in vitro fertilization (IVF) with fresh spermatozoa. The ROS treatment inhibited GV breakdown of 94.4% oocytes, with the majority arrested at the GV-I stage (67.4%). Maximum maturation rate to the metaphase-II stage after ROS treatment was achieved by 44 h of IVM (92.1%) and no differences were observed with control oocytes (95.0%). Penetration rate was correlated to the maturation rate. The duration of IVM had no effects on polyspermy and male pronuclear (MPN) formation rates at 8 h post insemination (hpi), whereas both rates increased at 22 hpi. Direct comparison with controls assessed at 22 hpi confirmed a lesser MPN formation in ROS-treated oocytes (73.7% compared with 53.6%). Glutathione (GSH) concentrations were less in oocytes treated with ROS than in control oocytes (5 compared with 7.7 pmol/oocyte) as well as blastocyst rate (22.0% compared with 38.1%, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pretreated with ROS for 48 h did not equal that of control oocytes in the current IVM system.

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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↗

PubMed2004

Reduction of the incidence of polyspermic penetration into porcine oocytes by pretreatment of fresh spermatozoa with adenosine and a transient co-incubation of the gametes with caffeine.

To reduce the incidence of polyspermic penetration, the effects of transient exposure of washed fresh spermatozoa to caffeine in a brief co-culture in vitro fertilization (IVF) system were examined. A pretreatment effect of spermatozoa with adenosine was also examined. When 5 mmol caffeine/l was supplemented during periods of co-culture and additional culture periods until 8 h after insemination, a shortened co-incubation period of gametes (30 denuded oocytes in 100 microl modified Medium 199-suspended spermatozoa at 2.5 x10(5) sperm/ml) from 30 to 5 min increased the monospermy rate in total mature oocytes examined. The number of spermatozoa binding to the zona surface was significantly lower in oocytes co-cultured for 5 min (33.1 +/- 2.2) than 8 h (207.6 +/- 13.7). A limited exposure of gametes to 5 mmol caffeine/l only during a transient co-culture period for 5 or 30 min significantly reduced the mean number of sperm cells that penetrated into the oocyte. Transient exposure of spermatozoa to caffeine for only 5 min increased the percentage of capacitated cells but not acrosome-reacted cells, as compared with a whole exposure treatment. Furthermore, preincubation of spermatozoa with 10 micromol adenosine/l for 90 min increased both the incidence of capacitated cells and the monospermy rate and consequently decreased the number of sperm cells that penetrated into the oocyte. In conclusion, these results have demonstrated that a new transient co-incubation IVF system, in which denuded oocytes are co-cultured with spermatozoa in medium containing caffeine for 5 to 30 min and then continuing the culture in caffeine-free medium, will reduce the incidence of polyspermic penetration. Preincubation of fresh spermatozoa with adenosine before the transient co-incubation IVF can also improve the monospermy rate. Furthermore, asynchrony in the morphology of sperm nuclei in polyspermic oocytes was reduced by the pretreatment with adenosine and a brief exposure to caffeine.

Adenosine↗

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.

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PubMedDate not supplied

Supplementation of the thawing media with reduced glutathione improves function and the in vitro fertilizing ability of boar spermatozoa after cryopreservation.

In this study, we evaluated the effects of glutathione (L-g-glutamyl-L-cysteinylglycine; GSH) supplementation of the thawing extender on semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To fully address these questions, we used a set of functional sperm tests. These included tests of motility and motion parameters, changes in sulfhydryl group content in membrane proteins, capacitation status, measures of intra-cellular reactive oxygen species generation, sperm chromatin condensation, and in vitro penetration of immature oocytes. The main findings emerging from this study were that addition of GSH to the thawing media resulted in a lower number of capacitated viable spermatozoa, a decrease in the number of spermatozoa with changes in the sulfhydryl groups in membrane proteins, a reduction of the reactive oxygen species generation, a lower chromatin condensation, and a higher penetration ability of oocytes in vitro and a higher proportion of decondensated sperm heads. GSH appears to play an important role in sperm antioxidant defense strategy. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen boar spermatozoa.

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