A. Chaveiro

Reactive Oxygen Species: A Double-Edged Sword in Reproduction

Antioxidants are compounds that protect cells against the damaging effects of reactive oxygen species (ROS) such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and peroxynitrite. Oxidative damage occurs to cells in vivo and in vitro from exposure to free radicals generated by exogenous agents (e.g., radiation, chemicals, hyperoxia) and endogenous processes such as normal cellular metabolism. An imbalance between antioxidants and ROS results in oxidative stress, leading to cellular damage. Oxidative stress (OS) has been linked to cancer, ageing, atherosclerosis, ischemic injury, inflammation, and neurodegenerative diseases. Under extreme oxidative conditions, or if the antioxidant protective mechanisms of cells are compromised, cellular injury and death may occur. Early mammalian embryos are susceptible to damage from reactive oxygen species, and they increase the production of oxygen free radicals when cultured in vitro. ROS generation results from mitochondrias oxidative phosphorylation. The electrons will leak from the inner mitochondrial membranes, being transferred by the oxygen molecule, resulting in an unpaired electron in the orbit. This leaking results in the generation of the superoxide molecules. ROS can also be generated by the cytoplasmic NADPH-oxidase, cytochrome p450 enzymes and the xanthine oxidoreductase enzymes. The excess of OS can have negative effects on the cellular environment and can result in impaired cellular growth in the embryo or apoptosis resulting in embryo fragmentation. Similarly to what happens in females, oxidative energy production in males is inevitably associated with the generation of ROS excessive concentrations of which can lead to cellular pathology. It has been established that ROS can function as signaling molecules and evidence is emerging that sperm may generate low and controlled concentrations of ROS, specifically O2-H2O2, as well as other species such as nitric oxide (NO), which, in his turn, act to mediate the processes of capacitation, hyperactivation and acrosome reaction crucial to the acquisition of fertilizing ability. In the present review the most important antioxidants and their mechanisms of action related to animal reproduction, are discussed.

Influence of Apoptosis in Bovine Embryo’s Development

The correlation between apoptosis and early bovine embryonic loss is still not fully elucidated. In the present study, the relationship between the arrest of bovine embryos at the different stages of development and apoptosis was evaluated. We used embryos 7 days after in vitro maturation and fertilization, and morphologic and biochemical apoptotic analyses were performed by using a phase contrast microscope and by the terminal transferase dUTP nick end-labelling respectively. For the statistic, the apoptotic cell ratio (ACR) was determined as the percentage of apoptotic cells per embryo. To evaluate the relation between ACR and fragmentation pattern, embryos were divided into five groups, groups I-V. To assess the relation between ACR and cytoplasmatic fragmentation, embryos were divided into three groups, according to the fragmentation percentage (<5%; 5-15% and >15%). Of the total 139 embryos included, 65 arrested at 2-8 cells; 14 arrested at 9-16 cells; 18 compacted morula and 42 were non-arrested blastocysts. The average number of embryonic fragmentation at different stages of the development, 2-8 cells, 9-16 cells, compacted morula and blastocyst, was 16.0 +/- 1.5, 28.7 +/- 4.4, 4.4 +/- 2.4 and 1 +/- 0.3 respectively. The embryos at the stage of arrested 9-16 cells and compacted morula had higher ACR than those at the blastocyst stage, excluding the stage of 2-8 cells (the genome is not yet active). The correlation detected between embryonic development and ACR was 0.92 (p < 0.01). It was observed that embryos possessing high fragmentation showed the higher ACR value (r = 0.98, p < 0.05). Comparing the results between fragmentation percentage and ACR, it was observed that the embryos with higher percentage of fragmentation corresponded to higher ACR (r = 0.97, p < 0.01). These results clearly demonstrated that bovine embryonic arrest at different stages of development is correlated with the apoptotic mechanisms.

Bovine Oocyte Quality in Relation to Ultrastructural Characteristics of Zona Pellucida, Polyspermic Penetration and Developmental Competence

In the present study, the effect of bovine oocyte quality related to ultrastructural characteristics of zona pellucida (ZP), polyspermic penetration and embryo developmental competence was evaluated. Cumulus-oocyte complexes were punctioned from 453 ovaries, classified as 1, 2, 3 and 4 according to their morphological aspect, matured for 24 h and then divided into two groups. In group A, oocytes were fixed in 2.5% glutaraldehyde and 0.1 m sodium cacodylate and examined under a scanning electron microscope. Photomicrographs were taken and ZPs pores were evaluated in squares of 6.4-microm width. In group B, oocytes were fertilized in vitro. After 48 h, non-cleaved oocytes were fixed for polyspermy evaluation. On days 7, 9 and 10, embryos were classified as developed (blastocysts and hatched blastocysts). Results showed that quality 1 oocytes revealed a ZP pore diameter of 0.50 +/- 0.07 microm, which was smaller than the observed on oocytes of quality 2 (0.83 +/- 0.10 microm), quality 3 (1.02 +/- 0.22 microm) and quality 4 (1.38 +/- 0.59 microm) (p < or = 0.05). For In Vitro Fertilization (IVF), results showed that embryos originating from oocytes classed as 3 and 4 had lower cleavage rate (68.4% and 43.8%) than those belonging to class 1 and 2 (79.5% and 69.3%) (p < or = 0.05). None oocyte classified as 3 and 4 developed to hatch blastocysts, while for oocytes belonging to quality 1 and 2, these values were, respectively, 15.2% and 12.5%. Concerning polyspermy, oocytes class 1 and 2 had lower polyspermic penetration than those belonging to class 3 and 4 (respectively 4.1%, 4.5%, 11.1% and 9.8%, for class 1, 2, 3 and 4). In conclusion, the present study demonstrated that oocytes with low qualities result in lower developmental competence and with high percentage of polyspermy after IVF, which can be the result of the ZP structure such as the number and the pores diameter.

Effects of plasma urea nitrogen levels on the bovine oocyte ability to develop after in vitro fertilization.

The overall aim of the present study was to evaluate in vitro development ability of oocytes recovered from 56 Holstein Frisian heifers with low [group 1 (G1): <13 mg /dl], moderate [group 2 (G2): 13-16 mg /dl] and high [group 3 (G3): >16 mg /dl] plasma urea nitrogen (PUN) concentrations, to determine whether PUN concentrations affect the competence of oocytes to progress to blastocysts after in vitro fertilization. In vitro oocyte and embryo development was assessed by blastocyst rates, embryo total cell numbers and apoptosis. Blood samples for the determination of PUN were collected 24 h prior to collection of the ovaries at the slaughter. A total of 112 ovaries were collected at a local abattoir and oocytes (n = 697) were aspirated, in vitro matured and fertilized. On day 8, blastocysts were assigned to the terminal dUTP nick end labelling assay. Cleavage rates were significantly higher (p < 0.001) for groups 1 and 2 than for group 3 (i.e. 72.5% and 72.2% vs 61.7%, respectively). The proportion of fertilized oocytes that developed into blastocysts was higher (p < 0.05) for group 1 than for group 3 (34.0% vs 23.0%, respectively). Day 8 blastocysts showed higher total cell counts (p < 0.05) for group 1 than for group 3 (123.7 vs 76.3), and a higher (p < 0.05) total apoptotic cell rate was found in group 3 (25.9 and 19.0 vs 43.2 for G1, G2 and G3, respectively). In conclusion, the ability of oocytes from heifers with increased levels of PUN to develop to the blastocyst stage was significantly reduced when standard routines for in vitro maturation, fertilization and culture were followed. These detrimental effects can be mediated in part through direct effect of urea and/or by the metabolic products on the process of follicle-enclosed oocyte nuclear and cytoplasmic development.

In vitro oocyte fertilization and subsequent embryonic development after cryopreservation of bovine ovarian tissue, using an effective approach for oocyte collection

This study was undertaken to assess dissection/puncture combined technique for collecting large number of oocytes from bovine ovaries and to determine the effect of ovarian tissue cryopreservation on the oocytes capability to undergo in vitro maturation, fertilization and subsequent embryonic development. Ovaries (n=31) of slaughtered cows were cut into small fragments using a scalpel blade and the ovarian tissues were randomly assigned to cryopreserved by slow freezing and vitrification and non cryopreserved (fresh) groups. Oocytes were collected from non-atretic follicles from fresh and post-thawing ovarian tissue by the puncture method. The advantage of this technique appeared through morphologically good quality cumulus-oocyte complex (COC) recovery rate from fresh tissue (31.7±2.0 oocytes/ovary). However, the cryopreservation affected the post thawing total and good quality COC recovery rates from slow freezing (26.6±2.0 and 23.5±2.3 oocytes/ovary, respectively) and vitrification groups (21.7±1.1 and 17.6±1.8 oocyte/ovary, respectively). The maturation rate resulted in significant differences between the fresh tissue (94.1±1.1%) and the two cryopreservation groups. Moreover, this rate was significantly higher in the slow freezing group (80.1±1.3%) than in the vitrification group (73.0±1.9%). No statistical differences were observed in the cleavage and the embryonic developmental rates between fresh tissue group and cryopreservation groups. Furthermore the number of embryos produced per animal was statistically higher for fresh tissues than for slow freezing and the vitrification groups (34.4±1.4, 27.8±3.1 and 22.0±0.7, respectively). In conclusion, dissection method followed by puncture of bovine ovaries greatly maximizes the number of good quality oocytes recovered, as well as the number of embryos obtained per animal. Ovarian tissue can be successfully cryopreserved by slow freezing and vitrification.

In Vitro Effect of the Reproductive Hormones on the Oxidative Burst Activity of Polymorphonuclear Leucocytes from Cows: A Flow Cytometric Study

In this study, the effect of reproductive hormones and substances with hormonal activity on the oxidative burst activity of blood polymorphonuclear leucocytes (PMN) high yielding dairy cows was evaluated. Different concentrations of: progesterone, oestradiol 17β, FSH, LH, GnRH, cortisol and PGF2α were incubated in vitro for 4 h with PMN of seven high milk yielding cows, during the period of anoestrous postpartum. Controls were run in parallel in which each hormone was replaced by its solvent. After incubation with hormones the competence of PMN to generate H(2) O(2) was monitored by flow cytometry. A down-regulation on the oxidative burst activity of PMA-stimulated PMN was observed when cells were incubated with progesterone. Significant (p ≤ 0.001) differences between control and progesterone incubated cells were observed from 6.56 μg/ml. The same predisposition was observed when PMNs were incubated with cortisol. Besides for all concentrations employed, a decrease in the burst activity was observed, only beyond 0.19 mg/ml, statistical differences between the results obtained by the control and the cortisol incubated cells were obtained. Concerning oestradiol 17β, an increase on H(2) O(2) -production was observed when PMN were incubated with 15 pg/ml and 45 pg/ml of this steroid (p ≤ 0.05), followed by a depression of the cells activity when unphysiological concentrations were employed. Significant (p ≤ 0.05) differences between the obtained with the control and oestradiol 17β incubated cells were observed only in the highest concentration of oestradiol. No statistical differences were observed in the metabolic burst activity of PMN incubated with FSH, GnRH and LH when compared with the results obtained by the control.

The effect of vitrification of immature bovine oocytes to the subsequent in vitro development and gene expression

Immature bovine oocytes were vitrified using the cryotop method and their post-warming survivability and capability to undergo in vitro maturation, fertilization and subsequent embryonic development were evaluated. In addition throughout the embryonic 2-cell, 4-cell, morula and blastocyst stages, the expression of four developmentally important genes (Cx43, CDH1, DNMT1 and HSPA14) was analysed using the real-time polymerase chain reaction (PCR). Immature oocytes (n = 550) were randomly assigned to non-vitrified (fresh) or cryotop vitrification groups using ethylene glycol (EG) with 1,2 propanediol (PROH) or dimethylsulphoxide (DMSO). After warming, oocytes survivability, embryo cleavage and embryonic developmental rates were not statistically different between the two cryoprotectants groups. However, the DMSO group had a lower (P < 0.05) oocyte maturation rate compared with the fresh and PROH groups. For morula and blastocyst rates, the DMSO group achieved a lower (P < 0.05) morula rate compared with the fresh group, while at the blastocyst stage, there were no differences between fresh and both cryoprotectants groups. For molecular analysis, at the 4-cell stage, most studied genes showed an inconsistent pattern of expression either from the PROH or DMSO groups. Noteworthily, these differences were limited at the morula and blastocyst stages. In conclusion, the cryotop method is sufficient for vitrification of immature bovine oocytes, both for embryonic developmental competence and at the molecular level. Moreover, PROH showed some advantage over DMSO as a cryoprotectant.

Optimisation of total RNA extraction from bovine oocytes and embryos for gene expression studies and effects of cryoprotectants on total RNA extraction

Gene expression is required for understanding bovine oocytes meiotic maturation as well as the potential of embryonic development. In the present study a standardized reagent protocol for total RNA extraction was designed for bovine oocytes and embryos, which is considered specific and less expensive. For such purpose oocytes (n = 795) recovered from about 80 ovaries were divided in three groups: Group 1 modified Trizol® (MTP, n = 355); Group 2 Guanidinium thiocyanate protocol (GNTC, n = 140) and Group 3 Commercial Kit protocol (CKP, n = 60). Oocytes belonging to group 1 (n = 100) and 3 (n = 20) were subjected to vitrification using two cryoprotectants 1,2 propandiol (PROH) or Dimethylsulfoxide (DMSO). The 240 remaining oocytes were divided into 3 groups in which 100 were used, in fresh, for in vitro fertilization, and 140 oocytes were vitrified using PROH (n = 70) and DMSO (n = 70) as cryoprotectants, being then fertilized in vitro after thawing. Embryos were used nine days after fertilization. Gene amplification (SDHA, GAPDH and DNMT1) was performed in oocytes, and gene quantification (DNMT1) in in vitro produced embryos at the stage of blastocyst (n ≈ 10). Efficiency of the extraction was further compared. The purity of all samples to different protocols ranged from 1.10 to 1.25 for GNTC protocol; from 2.05 to 2.63 for the CKP and from 1.50 to 2.11 for the developed MTP, being the last one nearest to the expected purity levels for RNA samples (1.7–2.0). On average, for 30 fresh oocytes, from spectrophotometer readings, total RNA concentration was 127.8 ± 9.3 ng μL−1 for MTP, against 46.4 ± 9.5 ng μL−1 from CKP and 47.6 ± 12.9 ng μL−1 for GNTC protocol. Using the MTP to evaluate RNA in 30 vitrified/thawed oocytes, resulted in a total RNA concentration of 61.3 ± 3.3 ng μL−1 and 40.0 ± 12.4 ng μL−1, respectively for DMSO and PROH. Regarding total RNA concentration and purity, in blastocyst stage, more purity was observed in DMSO as compared to PROH (1.8 vs. 1.2) (p < 0.05). Better results were also observed on the MTP for gene amplification when compared with the other protocols. For gene quantification, the proposed protocol quantified DNMT1 gene with PCR efficiency (0.933) after normalization against GAPDH and SDHA. Amplification and quantification of genes proved specificity and efficiency of the MTP over the other protocols.

Effect of α-tocopherol on in vitro maturation of bovine cumulus-oocyte complexes

This study determined: the effects of α-tocopherol on apoptotic and necrotic levels of cumulus cells after in vitro maturation of bovine oocytes; whether exposure to α-tocopherol facilitates the development of bovine enclosed oocytes to metaphase II; and the effects of this antioxidant on apoptotic and necrotic levels of granulosa cells cultured in vitro. In conclusion, supplementation with α-tocopherol on in vitro maturation of bovine oocytes has a detrimental effect on the ability of oocytes to reach metaphase II, increasing the number of apoptotic and necrotic cumulus cells of bovine cumulus oocyte complexes (COC). This antioxidant showed a slight improvement in the viability of cultured granulosa cells at a concentration of 100 µM. Key words: Bovine, oocyte maturation in vitro, antioxidant, α-tocopherol

Sire Effect and Sperm Apoptosis on Bovine Embryonic Cleavage and Subsequent In Vitro Embryo Development

The present study was conducted to evaluate sire effect on the kinetics of in vitro cleavage and further embryonic development after in vitro fertilization (IVF) using bulls of an Azorean rare breed called “Ramo Grande” (RG), and examine whether a relationship exists between bulls sperm apoptosis and sire in vitro fertility. Results showed that cleavage and blastocyst rates were statistically different between bulls (P<.05), being possible to notice different ability to produce embryos with good development competence (P<.05), as assessed by the proportion of fertilized oocytes that reached blastocyst stage. The proportion of blastocysts that continued the development to hatched blastocysts stage ranged from 9.1 to 87.5% (P<.05). Differences in sperm apoptosis increased during the swim-up procedure, which was statistically different (P<.05) between bulls, being negatively correlated with the ability of those bulls to fertilize oocytes and resulting embryos to develop to hatched blastocysts (R=−0.96, P≤.01). In conclusion, this study clearly demonstrated great differences between bulls, concerning apoptosis levels, which were negatively related to bulls ability to produce good quality embryos after IVF.