Marc-André Sirard

Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing.

Growing evidence suggests that the generation of reactive oxygen species (ROS) and their detoxification by antioxidants plays a very important role in fertility. However, the relationship between the level of antioxidants in spermatozoa and the decreased fecundity following a freeze/thaw cycle remains poorly understood. We assessed the activities of antioxidant enzymes such as catalase, glutathione peroxidase (GPx), superoxide dismutase (SOD), and levels of reduced/oxidized glutathione (GSH/GSSG) in bovine semen. Sperm cells were isolated using a Percoll gradient to avoid contamination from seminal plasma, cellular debris, and other cell types. We found that bovine spermatozoa are poorly adapted to metabolize the toxic hydrogen peroxide (H2O2). Indeed, very low levels of GPx and an absence of catalase were observed. We also studied the effect of freezing and thawing bovine spermatozoa in a egg yolk‐Tris‐glycerol extender (EYTG). Cryopreservation significantly reduced sperm GSH levels by 78% and SOD activity by 50%. We also investigated whether the decrease in GSH level could be linked to oxidative metabolism and found that a greater reduction in intracellular GSH level occurred when fresh sperm cells were incubated in EYTG for 6 hr at 38.5°C under aerobic conditions than when incubated under restricted oxygen availability. Our results strongly suggest the involvement of an oxidative stress during a freeze/thaw cycle and are consistent with the hypothesis that ROS generated during such a cycle are detrimental to sperm function. Mol. Reprod. Dev. 55:282–288, 2000.

Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen.

We previously showed that cryopreservation of bull spermatozoa in egg yolk Tris extender (EYTG) significantly reduced the intracellular level of thiols. Other studies showed the beneficial effects of adding antioxidants to cryopreserved bull spermatozoa. These studies led us to investigate the effects of various thiols, an important class of antioxidants, on sperm motility of cryopreserved bull semen in a commonly used extender, EYTG. Sperm motility was analyzed by computer-assisted semen analysis (CASA). After thawing, a diluted pool of bull semen was incubated at 38.5 degrees C in airtight tubes with the following thiols for 6 hours: glutathione (GSH/GSSG), cysteine, N-acetyl-L-cysteine (NAC) and 2-mercaptoethanol in the presence or absence of oxidative stress. The oxidative stress was caused by adding H2O2 (100 microM) to diluted semen. Incubation of diluted bull semen in EYTG at 38.5 degrees C over a period of 6 h decreased sperm motility by approximately 9 fold from the start (72 +/- 3, mean +/- SEM, n=4) to the end (9 +/- 4, n=4) of the incubation. We found that all thiols to a concentration above 0.5 mM maintained high sperm motility for 6 h in the absence of an external source of oxidative stress (52 +/- 4, for 4 thiols). However, one mM of each thiol was required to efficiently protect sperm motility in the presence of 100 microM of H2O2 for 6 h. We also found that the GSH concentration in diluted semen was too low (microM) to adequately supply exogenous addition of 72 U/mL of glutathione peroxidase (GPx), an enzyme that detoxifies H2O2 and hydroperoxides using GSH as a cofactor. In fact, a better protection of sperm motility could be achieved with only 5 U/mL of GPx and 0.1 mM of GSH added to diluted semen. Our results also demonstrated that added GSSG (0.5 mM) in diluted semen was not regenerated efficiently to GSH over 6 h. The latter result indicated in the extender that the glutathione redox-cycle was deficient. Therefore, deleterious effects sperm motility after cryopreservation in EYTG can be counteracted by adding various thiols at mM concentration.

Quantification of Housekeeping Transcript Levels During the Development of Bovine Preimplantation Embryos

Abstract In mammals, the study of gene expression in the preimplantation embryo has been difficult because the standard procedures used to quantify mRNA generally require large amounts of starting material. The development of protocols using different quantitative strategies generally involving the polymerase chain reaction (PCR) has provided new tools for exploration of gene expression in preimplantation embryos. However, the use of an internal standard, often referred as a housekeeping gene, is essential to normalize the mRNA levels. RNA levels of eight housekeeping genes were quantified using real time PCR throughout the preimplantation period of the bovine embryo to find the most suitable gene to be used as standard. Histone H2a was the best internal standard because the transcript levels were constant across the preimplantation period. Linear amplification of antisense RNA using the T7 promotor for in vitro transcription of the entire RNA pool was evaluated as a suitable way to preamplify the starting material prior to quantification and was effective in providing accurate RNA abundance profiles throughout the preimplantation period. However, the amplification appears to be template dependent because the amplification factors were higher for some genes.

Manipulation of Follicular Development to Produce Developmentally Competent Bovine Oocytes

Abstract Superstimulation in donor cows increases the number of cumulus-oocyte complexes (COC), but when compared to in vivo maturation, in vitro maturation results in only half as many blastocysts after prolonged in vitro culture. The objective of this study was to establish a superstimulation protocol that would produce a maximal number of competent COC for standard in vitro embryo production. During experiment 1, eight cyclic Holstein heifers were superstimulated with four doses of FSH. Half the heifers received an injection of LH 6 h before ovum pick-up (OPU). The COC were collected following OPU either 33 or 48 h following the last FSH injection (coasting period). During experiment 2, six cyclic Holstein heifers were superstimulated with six doses of FSH, and in half the heifers, LH was administered 6 h before OPU. The COC were collected following ultrasound-guided transvaginal aspiration of both ovaries 48 h after the last FSH injection (coasting period). The COC originating from follicles with a diameter of 5 mm or more (n = 180 for experiment 1 and 57 for experiment 2) were subjected to standard in vitro maturation, fertilization, and development. When animals were administered four doses of FSH, 48 h of coasting resulted in significantly more 5- to 10-mm follicles (P < 0.01) than 33 h of coasting. If a 33-h coasting period was used, administration of LH 6 h before OPU resulted in a significant increase in both percentage of blastocysts and embryo production rate at Days 7 and 8 (P ≤ 0.05) of in vitro culture. If a 48-h coasting period was used, LH injection did not affect the rates of blastocyst production. When donors were administered six doses of FSH with a 48-h coasting period, the highest results, although not significant (P < 0.08), were obtained when animals received LH 6 h before OPU, with 80% ± 9% (mean ± SEM) blastocysts and 0.8 ± 0.09 embryo produced per COC retrieved per heifer at Day 8 of culture. Never has in vitro technology been so close to producing 100% developmentally competent COC.

Identification of differentially expressed markers in human follicular cells associated with competent oocytes

BACKGROUND The development of an accurate method for selection of high-quality embryos is essential to achieve high pregnancy rates with single embryo transfer in human IVF. The developmental competence of the oocyte is acquired during follicle maturation and strong communication also exists between the follicular cells (FCs) and the oocytes; thus oocyte developmental competence may be determined by markers expressed in the surrounding FCs. METHODS From consenting patients (n = 40), FCs were recovered on a per follicle basis by individual follicle puncture. Hybridization analyses using a custom-made complementary DNA microarray containing granulosa/cumulus expressed sequence tags (ESTs) from subtracted libraries and an Affymetrix GeneChip were performed to identify specific genes expressed in follicles leading to a pregnancy. The selected candidate genes were validated by quantitative-PCR (Q-PCR). RESULTS Subtractive libraries prepared from pooled samples representing pregnant versus non-pregnant patients produced 1694 ESTs. Hybridization data analysis discriminated 115 genes associated with competent follicles. Selected candidates were confirmed by Q-PCR: 3-beta-hydroxysteroid dehydrogenase 1 (P = 0.0078), Ferredoxin 1 (P = 0.0203), Serine (or cysteine) proteinase inhibitor clade E member 2 (P = 0.0499), Cytochrome P450 aromatase (P = 0.0359) and Cell division cycle 42 (P = 0.0396). CONCLUSIONS Microarray technologies are useful to mine the transcriptome of FCs expressed in follicles associated with competent oocytes and could be used to improve embryo selection with the objective of successful single embryo transfer.

IN VITRO PRODUCTION OF BOVINE EMBRYOS : DEVELOPMENTAL COMPETENCE IS ACQUIRED BEFORE MATURATION

Few studies have examined the importance of the time during which oocytes are left in the ovaries following animal slaughter. The objective of this study was to determine the optimal time for retrieving oocytes after slaughter and to ascertain if superovulating cows in association with this optimal time could increase the developmental competence of bovine oocytes. In Experiment 1, oocytes were left in the postmortem ovaries for 2,3,4,5,6 or 7 h and were then transported to the laboratory at approximately 30 degrees C. Recovered oocytes were processed in vitro using standard techniques. In Experiment 2, cyclic heifers (n = 18) were superovulated between Days 8 and 12 of the estrous cycle with 8 constant doses (4 mg each, twice daily) or 8 decreasing doses (2 injections of 4,3,2 and 1 mg every 12 h) of FSH-P +/- 1 mg prostaglandin 24 or 48 h before slaughter. Oocytes were left in the ovaries for 4 h and were classified according to the state of their cumulus and cytoplasm. The results indicated that oocytes aspirated from ovaries collected 4 h after slaughter produced significantly more > or =64-cell embryos after 7 d of in vitro development than those collected 2, 6 or 7 h postslaughter. Oocytes (87%) from superovulated animals had numerous layers of cumulus cells and originated from medium (2.7 to 8 mm) and large (> or =8 mm) follicles. Significantly more oocytes developed from large follicles than from medium follicles. Although individual culture of the oocytes negatively affected the percentage of embryos produced, group culture of oocytes from animals that were superovulated and left in the postmortem ovaries for 4 h resulted in exceptionally high rates of embryos after 5 d of IVD. On average, 60 to 80% of 16-cell embryos were produced, indicating that under the proper conditions, developmental competence is acquired before in vitro maturation.

Antioxidant requirements for bovine oocytes varies during in vitro maturation, fertilization and development

Antioxidants may be beneficial additives to synthetic culture media because these well defined media lack serum or other macromolecules that serve as reactive oxygen species scavengers. In this study, three separate experiments were performed to determine the effects of antioxidants on the development of oocytes to the morula and blastocyst stage when added during in vitro maturation (IVM) of bovine oocytes, during in vitro fertilization (IVF), and during embryo culture for the first 72 h of the development period. Bovine oocytes were matured, fertilized (under 20% O(2)), and embryos were cultured (under 7% O(2)) in defined conditioned medium in vitro with or without supplementation with the antioxidant cysteine, N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD). Significant improvements in the proportion of oocytes undergoing morula and blastocyst development (33.3% versus 20.3%, P<0.05) were achieved when cysteine (0.6 mM) was added to the maturation medium as compared to control medium without antioxidant supplementation. However, the addition of NAC (0.6mM), catalase (5 or 127 U/ml) or SOD (10 or 1000 U/ml) to the maturation medium did not improve the proportion of oocytes undergoing morula and blastocyst development. During the IVF period, addition of antioxidants (cysteine or NAC 0.6mM, catalase 127U/ml, SOD 100U/ml) significantly reduced the subsequent rate of bovine embryo development to the morula and blastocyst stage (P<0.05). In a defined medium for embryo culture (7% O(2)), the addition of cysteine improved the development of bovine embryos while NAC, catalase and SOD had no positive effect on embryonic development. Our study showed that medium supplementation with cysteine during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like catalase and SOD that caused no improvement.

Identification of Potential Markers of Oocyte Competence Expressed in Bovine Cumulus Cells Matured with Follicle-Stimulating Hormone and/or Phorbol Myristate Acetate In Vitro

Abstract Oocyte competence is the ability of the oocyte to complete maturation, undergo successful fertilization, and reach the blastocyst stage. Cumulus cells are indispensable for this process. Their removal significantly affects the blastocyst rates. Moreover, the properties and functions of cumulus cells are regulated by the oocyte. They also reflect the oocytes degree of maturation. Our study was aimed at identifying markers of oocyte competence that are expressed in bovine cumulus cells. In a previous study in our laboratory, the blastocyst yield following FSH or phorbol myristate acetate (PMA) treatment was 45%%. Therefore, we tested four sets of conditions during the first 6 h of in vitro maturation (IVM): FSH (0.1 μg/ml), PMA (0.1 μM), FSH ++ PMA, and negative control. Extracts from each IVM treatment were hybridized against the same negative control on a microarray containing a partial library of differentially expressed transcripts in the cumulus of competent oocytes collected at 6 h after LH in vivo. Common positive clones between diffentially treated cells were selected, and 15 candidates were validated by real-time PCR. Based on this, the main candidates expressed in cumulus cells and that could be valuable and indirect markers of oocyte competence are hyaluronan synthase 2 (HAS2), inhibin βA (INHBA), epidermal growth factor receptor (EGFR), gremlin 1 (GREM1), betacellulin (BTC), CD44, tumor necrosis factor-induced protein 6 (TNFAIP6), and prostaglandin-endoperoxide synthase 2 (PTGS2). These biomarkers could be potential candidates to predict oocyte competence and to select higher-quality embryos for transfer. Additionally, these indirect predictors of oocyte competence and follicular health could improve our knowledge of gene expression patterns in the cumulus and yield insights into the molecular pathways controlling oocyte competence.

Effect of the Absence or Presence of Various Protein Supplements on Further Development of Bovine Oocytes During In Vitro Maturation

Abstract The evaluation of culture medium for bovine oocytes has progressed toward more defined conditions during the last few years. The main objective of this study was to evaluate different sources of albumin as a protein supplement during in vitro maturation (IVM) of bovine oocytes in synthetic oviduct fluid medium (SOF). The replacement of protein with polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA) was also evaluated. The effect of recombinant human FSH on cumulus expansion and nuclear maturation in SOF containing BSA (BSA-V) or PVP-40 was also studied. Addition of BSA-V during IVM retarded nuclear maturation when compared with addition of PVP-40 or use of SOF alone. The inclusion of different concentrations of BSA-V, fetal calf serum (FCS), or PVA during IVM had no positive effect on the developmental capacity of the oocytes compared with the use of SOF alone with no supplement but significantly decreased the percentage of embryos reaching the morula and blastocyst stages. However, when BSA-V was replaced with purified BSA, BSA that was essentially free of fatty acids, or chicken egg albumin, embryonic development rates were restored. The presence of PVP-40 but not PVP-360 during IVM significantly increased morula and blastocyst production. These results indicate that although SOF alone can support bovine oocyte maturation, a high proportion of morulae and blastocysts can be produced from IVM oocytes cultured in medium containing PVP-40. These studies are the first to show that the effect of FSH on nuclear maturation and cumulus expansion is dependent on substrates present in IVM medium.

Oocyte maturation and IVF in cattle

Abstract There is still a clear difference between ova obtained from in vivo maturation and oocytes matured in culture. Eggs obtained following the LH surge in vivo show a higher potential for development. These observations indicate that cytoplasmic competence must be different between the in vitro and the in vivo matured oocytes. Besides these functional differences, a few studies have revealed additional discrepancies between in vitro and in vivo maturation at the ultrastructure level. Differences in terms of subsequent anomalies were also observed at the chromatin level in morulae-blastocysts obtained with either procedure. There are two possible explanations for the differences observed: the culture conditions used or the initial intrinsic competence of the oocyte. It is certainly feasible to improve the culture conditions during maturation, but after 10 years of collective efforts, it seems that a specific signal or component is missing rather than inadequate or toxic culture conditions. If the immature oocytes possess differing degrees of competence, these might be due to factors affecting the oocyte during late folliculogenesis. Different follicular conditions would lead to differing oocyte competence. The origins of the oocytes are discussed with respect to five different aspects: cumulus morphology, follicular size, follicular health, ovarian stimulation and the oocyte handling procedure before the beginning of incubation. Our results, in addition to the evidence from the literature, suggest that the oocyte enters a permissive state when obtained from large and differentiated follicles as in dominance, early atresia or near ovulation. Developmental competence can then be induced by either LH in vivo or mimicked by an exposure of the ovary to warm incubation conditions for a few hours before collecting the oocytes. The increased development obtained in these conditions suggest that the ability to continue to the blastocyst stage is acquired in the follicle.