María Elena Arias

Evaluation of different culture systems with low oxygen tension on the development, quality and oxidative stress-related genes of bovine embryos produced in vitro

The present study was conducted to assess the development, quality and gene expression profile of oxidative stress-related genes of bovine embryos cultured in different culture systems with low oxygen tension (5% CO2, 5% O2 and 90% N2). The systems assessed included: (1) an incubator chamber; (2) a plastic bag; and (3) a foil bag. The choice of culture system had no effect on cleavage rate at 72 h. However, significant differences (P < 0.01) were observed in the rate of blastocysts registered at day 7 (29.8, 20.2 and 12.7% for incubator chamber, plastic bag and foil bag, respectively). Total number of cells did not differ between systems, although the proportion of ICM:total cells was affected particularly in the plastic bag (19.5%), compared with the incubator chamber (31.4%). In addition, significant differences were found in the apoptotic:total cell ratio (3.3, 6.5 and 8.8% for the incubator chamber, plastic bag and foil bag, respectively), with apoptotic nuclei localised mainly in the ICM compartment of the embryo. The amount of reactive oxygen species was also different between culture systems and this effect was correlated with a higher expression of SOD2, GSS and GPX1 genes in embryos cultured in the gassed bags as compared with embryos cultured in the incubator chamber. In conclusion, these results give evidence that, under low oxygen tension, the incubator chamber is more efficient and generates higher number of, and better quality, embryos than gassed bag systems evaluated here and this effect was probably due to an increased level of reactive oxygen species in the gassed bags, which upregulates the expression of some antioxidant enzymes to compensate for hyperoxia conditions.

Stability of reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) data in bovine blastocysts produced by IVF, ICSI and SCNT.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive and accurate tool for quantitative estimation of gene transcription levels in preimplantation embryos. To control for possible experimental variations, gene expression data must be normalized using internal control genes commonly known as reference genes. However, the stability of reference genes can vary depending on the state of development and/or experimental conditions; hence the assessment of their stability is essential before initiating a gene expression analysis. In the present study, we used RT-qPCR to measure the transcript levels of 10 commonly used reference genes and analyzed their expression stability in bovine blastocysts produced by in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). Using the geNorm program, we found the best combination of genes to normalize gene expression data in bovine embryos at the blastocyst stage produced by IVF (HMBS, SF3A1, and HPRT1), ICSI (H2A, HMBS, and GAPDH), SCNT (ACTB, SF3A1, and SDHA) and/or between blastocysts produced by these methods (GAPDH, HMBS and EEF1A2). We also demonstrated that not only the culture conditions may affect the expression patterns in bovine blastocysts but also the choice of embryo production method may have an important effect.

Effect of sperm pretreatment with sodium hydroxide and dithiothreitol on the efficiency of bovine intracytoplasmic sperm injection

The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is lower than in other species due, in part, to a lack of optimal conditions for its implementation; this has hindered the achievement of high rates of embryonic development and the birth of live offspring. The aim of the present study was to evaluate the effects of pretreatment of bovine spermatozoa with NaOH and dithiothreitol (DTT) on the viability, plasma membrane integrity, DNA fragmentation and in vitro developmental potential of embryos generated by ICSI. Following pretreatment of spermatozoa with 5 mM DTT for 20 min and a low concentration of NaOH (1 mM for 60 min), there were fewer live and acrosome reacted spermatozoa (44% and 34%, respectively) than in the control group without treatment (82%). Spermatozoa subjected to higher alkali concentrations (10-50 mM) were mostly dead and reacted. However, pronuclear formation, cleavage, blastocyst rate and embryo quality did not differ between these pretreatment groups and the untreated control group. In conclusion, we have described, for the first time, the effects of NaOH treatment on bovine spermatozoa and subsequent in vitro embryonic development after ICSI, and have demonstrated that pretreatment of bovine spermatozoa with NaOH or DTT is not necessary for an appropriate in vitro embryo development in this species.

Intracytoplasmic sperm injection affects embryo developmental potential and gene expression in cattle

Some reports have linked intracytoplasmic sperm injection (ICSI) with chromosomal abnormalities, low developmental potential and altered gene expression in embryos. ICSI has also been linked with obesity, early aging and increased incidence of tumors in offspring. Other reports have demonstrated that some of these complications disappeared within a few weeks of life or even showed a lack of such associations. The aim of this study was to evaluate and compare embryo development, quality and gene expression in bovine embryos generated by ICSI and by conventional in vitro fertilization (IVF) insemination. The results showed differences in cleavage (88.5% in IVF and 64.1% in ICSI) and blastocyst formation rates (36.1% in IVF and 22.3% in ICSI). The proportion of ICM cells to total cell count was higher in ICSI (39.2%) than in IVF embryos (29.5%). However, no differences were observed in the total embryonic cell numbers (159.3±28.5 and 161.2±56.2 for IVF and ICSI, respectively) or in the proportion of apoptotic nuclei to the total embryonic cell numbers (2.12 and 2.64% for IVF and ICSI, respectively). Gene expression analysis showed a down-regulation of insulin-like growth factor 2 (IGF2) and overexpression of bcl-2-like protein 4 (BAX), octamer-binding transcription factor four (OCT4), interferon-tau (IFNt), Mn-superoxide dismutase in the mitochondria (SOD2), and catalase (CAT) in embryos generated by ICSI. In conclusion, our study demonstrated differences in the morphological development of bovine embryos as well as in the expression of genes involved in early development between ICSI and IVF embryos. The results may indicate lower developmental potential of ICSI embryos compared with that of IVF.

Improved preimplantation development of bovine ICSI embryos generated with spermatozoa pretreated with membrane-destabilizing agents lysolecithin and Triton X-100

In cattle, intracytoplasmic sperm injection (ICSI) has a low efficiency. The acrosome content may be responsible for this effect because of the large amount of hydrolytic enzymes that are released within the oocyte. With the aim of removing the acrosome and destabilize the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX) at different concentrations. We evaluated the membrane integrity, the acrosome integrity, DNA integrity, and the variation of phospholipase C zeta. The rates of development (cleavage and blastocysts) were also evaluated along with pronuclear formation and the embryo quality. Spermatozoa incubated with LL and TX (0.01%, 0.02%, 0.03%, and 0.04%) decreased (Pxa0<xa00.0001) sperm viability in a dose-dependent manner. The acrosome reaction was also increased (Pxa0<xa00.0001) in all tested concentrations of LL and TX achieving 100% at 0.05% concentration in both treatments. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay reported an increase (Pxa0<xa00.05) in DNA fragmentation only with the highest concentration of LL (0.06%), whereas all concentrations assessed of TX reported an increased respect to the control. Phospholipase C zeta expression decreased (Pxa0<xa00.05) in spermatozoa treated with LL and TX at all concentrations tested. A higher cleavage rate was observed in ICSI-TX (66%) and ICSI-LL (65%) groups compared with the untreated control group (51%) and the blastocyst formation rate significantly increased in the ICSI-LL group (29%) compared with the control (21%). No differences were observed in the pronuclear formation and quality of the embryos. In conclusion, the destabilization of the plasma membrane and the release of the acrosomal content with LL and TX before ICSI improve the rate of embryonic development, without affecting the quality of the embryos produced by this technique.

Methyl-β-Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida-Binding Ability of Frozen/Thawed Bovine Spermatozoa.

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein-free media. However, the additive capacitating effect of methyl-β-cyclodextrin (MβCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen-thawed bovine spermatozoa in a BSA-containing medium supplemented with MβCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MβCD in a dose-dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MβCD. However, pre-incubation of spermatozoa in MβCD-supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)-binding ability (p < 0.05). Thus, we conclude that MβCD supplementation is able to enhance the capacitation status of frozen-thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.

Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT) embryos.

Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01) in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

Supplementation of IVF medium with melatonin: effect on sperm functionality and in vitro produced bovine embryos

Gamete co‐incubation generates high free radical levels surrounding growing zygotes which may impair subsequent embryo viability. Melatonin eliminates a wide variety of free radicals; hence, we tried to improve in vitro embryo production by adding melatonin to in vitro fertilisation (IVF) media in high (Exp. 1) and low concentrations (Exp. 2), and we evaluated its effect on bull sperm function during IVF co‐incubation time (Exp. 3). In Experiment 1, we supplemented IVF media culture with 0.01, 0.1 and 1 mmol of melatonin, along with a no melatonin control group. In Experiment 2, melatonin levels were reduced to 10, 100 and 1000 nmol, with a no melatonin control group. In Experiment 3, spermatozoa were incubated in IVF media with melatonin (as Exp. 2) and functional parameters were analysed at 0, 4 and 18 h. In Experiment 1, only 1 mmol melatonin showed lesser blastocyst rates than control (C: 23.2 ± 6.7% versus 1 mmol: 2.0 ± 1.7%). In Experiment 2, no statistical differences were found in cleavage percentage, blastocyst percentage and total cell count for any melatonin treatment. In Experiment 3, sperm samples with 1000 nmol melatonin had a significantly higher wobbler (WOB) coefficient, a lower percentage of intact acrosomes, a lower percentage of viable spermatozoa with ROS, greater DNA fragmentation and higher DNA oxidation than controls. Total fluorescence intensity for ROS at 10 nmol melatonin was significantly greater than controls (P < 0.05). IVF media with 1 mmol melatonin is deleterious for embryo development, and in lower concentrations, it modulated sperm functionality, but had no effects on embryo production.

Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer.

Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

Sperm capacitation pretreatment positively impacts bovine intracytoplasmic sperm injection

The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is low compared to other species due in part to inadequate egg activation and sperm nucleus decondensation after injection. We hypothesized that this low efficiency is due to the lack of complete sperm capacitation, so we evaluated the effects of isobutylmethylxanthine (IBMX) and methyl‐β‐cyclodextrin (MβCD) on bovine sperm capacitation and on the preimplantation developmental potential of bovine embryos generated by ICSI. Treatment with IBMX and MβCD decreased sperm viability (between 13–30%); nevertheless, 0.4u2009mM IBMX and 1u2009mM MβCD increased (pu2009<u20090.05) capacitation metrics—that is, acrosome exocytosis, intracellular calcium level, plasma membrane fluidity, and tyrosine phosphorylation—compared to the control. After ICSI, embryos injected with IBMX‐ and MβCD‐treated sperm showed similar cleavage to the untreated group (range 82–88%). Pronucleus formation rate was higher with MβCD‐pretreatment (54%) compared to the control group (25%), and blastocyst rate was significantly improved with MβCD‐pretreatment (24%) compared to the IBMX (18%) and control (17%) groups. Importantly, embryo quality—as assessed by the total number of cells, cell allocation, and apoptotic cell index—was not affected by the sperm treatments. In conclusion, MβCD pretreatment of sperm improved the efficiency of blastocyst production in bovine ICSI.