Miriam Castillo-Martín

Comparative effects of adding β-mercaptoethanol or L-ascorbic acid to culture or vitrification–warming media on IVF porcine embryos

The aims of the present study were to; (1) determine the effects of supplementation with two antioxidants during in vitro culture (IVC) on embryo development and quality; and (2) test the effects of adding the antioxidants to vitrification-warming media on the cryotolerance of in vitro-produced (IVP) porcine blastocysts. In Experiment 1, presumptive zygotes were cultured without antioxidants, with 50 µM β-mercaptoethanol (β-ME) or with 100 µM L-ascorbic acid (AC). After culture, blastocyst yield, quality and cryotolerance were evaluated in each treatment group. In Experiment 2, survival rates (3 and 24 h), total cell number, apoptosis index and the formation of reactive oxygen species (ROS) in blastocysts vitrified-warmed with 100 µM AC or 50 µM β-ME or without antioxidants added to the vitrification medium were compared. Antioxidant addition during IVC had no effect on embryo development, total cell number or the apoptosis index, and culturing embryos in the presence of β-ME had no effects on cryotolerance. In contrast, ROS levels and survival rates after vitrification-warming were significantly improved in embryos cultured with AC. Furthermore, addition of AC into vitrification-warming media enhanced embryo survival and embryo quality after warming. In conclusion, our results suggest that supplementing culture or vitrification media with 100 µM AC improves the quality and cryosurvival of IVP porcine blastocysts.

Direct binding of boar ejaculate and epididymal spermatozoa to porcine epididymal epithelial cells is also needed to maintain sperm survival in in vitro co-culture

The aim of the present study was to compare the influence of cultured epididymal epithelial cells (EEC) from corpus, caput or cauda, oviductal epithelial cells (OEC) and non-reproductive epithelial cells (LLC-PK1) on function and survival of epididymal and ejaculated spermatozoa, in the latter case to determine whether such influence differed between morphologically normal and abnormal spermatozoa. For this purpose, either spermatozoa were directly co-cultured with EEC from caput, corpus, or cauda, OEC and LLC-PK1 cells (experiment 1) or a membrane-diffusible insert was included in these co-cultures (experiment 2). EEC cultured from the three epididymal regions did not differently affect the sperm parameters. Morphologically normal spermatozoa presented a higher ability to bind EEC, OEC, and LLC-PK1 than abnormal spermatozoa with cytoplasmic droplets or with tail/head malformations. Epididymal spermatozoa were more able to bind EEC during the first 24 h of co-culture, while ejaculated spermatozoa presented a higher capacity to bind OEC between 30 min and 3 h of co-incubation. In all cases, the ability to bind to epithelial cells was higher when they were co-cultured with EEC and OEC than with LLC-PK1. After 2 h of co-culture, the viability of epididymal spermatozoa was better maintained when they bound EEC than when they bound OEC. Conversely, the viability of ejaculated spermatozoa was better maintained when bound OEC than when bound EEC after 24 and 48 h of co-culture. Our work, apart from corroborating the involvement of morphologically normal spermatozoa in the formation of sperm reservoir, highlights the importance of direct contact spermatozoa-EEC in maintaining the sperm survival in in vitro co-culture, and also suggests that a specific binding between EEC and epididymal spermatozoa exists.

Cryotolerance of in vitro-produced porcine blastocysts is improved when using glucose instead of pyruvate and lactate during the first 2 days of embryo culture.

The objective of the present study was to determine the effects of replacing glucose with pyruvate and lactate during the first 48 h of in vitro culture (IVC) in NCSU-23 medium on embryo development, embryo quality and survival of porcine blastocysts after vitrification. To this end, in vitro-produced (IVP) porcine oocytes were cultured with either glucose for 6 days (IVC-Glu) or pyruvate-lactate from Day 0 to Day 2 and then with glucose until Day 6 (IVC-PyrLac). Blastocysts were vitrified on Day 6 using the Cryotop device and, after warming, survival rate and the apoptosis index were evaluated after 24 h incubation in NCSU-23 medium. No significant differences were observed between IVC-Glu and IVC-PyrLac in terms of cleavage rate, blastocyst yield, total number of cells per blastocyst or the apoptosis index (1.82±0.75% vs 3.18±0.88%, respectively) of non-vitrified embryos. However, a significant increase was seen in hatching/hatched blastocysts in the IVC-PyrLac compared with IVC-Glu treatment group (12.71±1.20% vs 3.54±0.47%, respectively). Regardless of treatment, vitrification impaired the survival rate and the apoptosis index. When comparing both treatments after warming, the percentage of apoptotic cells was significantly higher for blastocysts in the IVC-PyrLac compared with IVC-Glu group (18.55±3.49% vs 9.12±2.17%, respectively). In conclusion, under the conditions of the present study, replacement of glucose with pyruvate-lactate during the first 48 h of culture resulted in a lower cryotolerance of IVP porcine embryos.

Supplementing culture and vitrification-warming media with l-ascorbic acid enhances survival rates and redox status of IVP porcine blastocysts via induction of GPX1 and SOD1 expression

The present study sought to determine the effect of adding l-ascorbic acid (AC) to (1) in vitro culture medium and (2) vitrification and warming solutions on redox status and developmental ability and quality of IVP porcine embryos. In both experiments, embryo quality was analysed in terms of total cell number (TCN), DNA fragmentation, intracellular peroxide levels and expression of three oxidative stress-related genes: glutathione peroxidase 1 (GPX1), superoxide dismutase 1 (SOD1) and 2 (SOD2). In the first experiment, fresh blastocysts were found to upregulate SOD1 expression when cultured with medium supplemented 100 μM AC. No differences were found between culture groups in the other analysed parameters. In the second experiment, blastocysts cultured with or without AC were divided into two groups: vitrified and warmed with solutions containing 0 or 100 μM AC. Addition of AC during culture and vitrification-warming upregulated the expression of GPX1 and SOD1 genes, enhanced survival rates and decreased peroxide levels at 24h post-warming. In addition, peroxide levels were negatively correlated with relative GPX1- and SOD1-transcript abundances, whereas GPX1 was positively correlated with embryo survival at 24h post-warming. No effects of AC-supplementation were seen for TCN, DNA fragmentation or relative SOD2-transcript abundance in vitrified blastocysts. In conclusion, the addition of AC to culture and vitrification-warming media increases gene expression of antioxidant enzymes SOD1 and GPX1. This appears to improve redox balance and is suggested to ultimately enhance embryo cryosurvival.

Triosephosphate isomerase (TPI) and epididymal secretory glutathione peroxidase (GPX5) are markers for boar sperm quality

The present study sought to determine the relationship of three sperm proteins (acrosin binding protein, ACRBP; outer dense fibre protein 1, ODF1; and triosephosphate isomerase, TPI), and two seminal plasma proteins (fibronectin, FN1; and epididymal secretory glutathione peroxidase, GPX5) to conventional sperm quality parameters (sperm membrane integrity, morphology and motility) in pigs. With this purpose, 22 boar ejaculates were split into two groups according to their sperm quality (mean±standard error of the mean, % viable sperm: 95.25±0.53 vs. 78.22±1.93; % morphologically normal sperm: 96.30±0.66 vs. 80.81±2.28). The amounts of these five proteins were evaluated through Western blot analysis and subsequently compared between these two groups through a t-test for independent samples. Normalised levels of TPI in sperm were significantly higher in the low than in the high sperm quality group. In addition, TPI was found to be negatively correlated with sperm membrane integrity, morphology and several parameters describing sperm motility. On the other hand, amounts of GPX5 in seminal plasma were also significantly higher in the low than in the high quality group, and this protein was also found to be negatively correlated with sperm membrane integrity and total and progressive sperm motility. By contrast, sperm content in ACRBP and ODF1 amounts of seminal plasma protein FN1 did not significantly differ between the two groups of sperm quality. Thus, we can conclude that sperm TPI content and amounts of GPX5 in seminal plasma may be used as quality markers of boar sperm.

Effects of vitrification on the expression of pluripotency, apoptotic and stress genes in in vitro-produced porcine blastocysts

The aims of the present study were to: (1) evaluate the effect of vitrification and warming on quality parameters and expression levels of pluripotency, apoptotic and stress genes in in vitro-produced (IVP) porcine blastocysts; and (ii) determine the correlation between these parameters. To this end, total cell number, DNA fragmentation, peroxide levels and the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), heat shock protein 70 (HSPA1A), POU class 5 homeobox 1 (POU5F1), superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) were analysed in fresh and vitrified IVP blastocysts. The results suggest that vitrification procedures have no effect on total cell number and gene expression of BAX, BCL2L1, SOD1 and SOD2 or the BAX:BCL2L1 ratio. Nevertheless, a significant increase in DNA fragmentation (2.9±0.4% vs 11.9±2.0%) and peroxide levels (80.4±2.6 vs 97.2±3.1) were seen in vitrified compared with Day 7 fresh blastocysts. In addition, after blastocyst vitrification, relative transcript abundance was downregulated for POU5F1 and upregulated for HSPA1A. Finally, there was a significant correlation of POU5F1 and HSPA1A with DNA fragmentation (POU5F1, r=-0.561; HSPA1A, r=0.604) and peroxide levels (POU5F1, r=-0.590; HSPA1A, r=0.621). In conclusion, under the conditions of the present study, vitrification and warming of IVP porcine blastocysts resulted in altered expression of POU5F1 and HSPA1A, but had no effect on BAX, BCL2L1, SOD1 and SOD2 expression.

Cryotolerance of porcine in vitro-produced blastocysts relies on blastocyst stage and length of in vitro culture prior to vitrification

The aim of our study was to assess whether the cryotolerance of in vitro-produced embryos could be influenced by the length of in vitro culture and size of blastocoel cavity before vitrification, using the pig as a model. For this purpose we analysed the cryoresistance and apoptosis rate of blastocysts at different stages of development as derived on Day 5 and 6 of in vitro culture. Blastocysts were subsequently vitrified, warmed and cultured for 24h. Re-expansion rates were recorded at 3 and 24h and total cell number and apoptotic cells were determined at 24h. Day-6 blastocysts showed the highest rates of survival after warming, which indicates higher quality compared with Day-5 blastocysts. Higher re-expansion rates were observed for expanded blastocysts and those in the process of hatching when compared with early blastocysts. Total cell number and apoptotic cells were affected by blastocyst stage, vitrification-warming procedures and length of in vitro culture, as expanding and hatching-hatched blastocysts from Day 6 presented higher percentages of apoptotic cells than fresh blastocysts and blastocysts vitrified at Day 5. Our findings suggest that the cryotop vitrification method is useful for the cryopreservation of porcine blastocysts presenting a high degree of expansion, particularly when vitrification is performed after 6 days of in vitro culture. Furthermore, these results show that faster embryo development underlies higher blastocyst cryotolerance and provide evidence that blastocoel cavity expansion before vitrification is a reliable index of in vitro-produced embryo quality and developmental potential.

Addition of L-ascorbic acid to culture and vitrification media of IVF porcine blastocysts improves survival and reduces HSPA1A levels of vitrified embryos.

The aim of the present study was to determine the effect of L-ascorbic acid on embryo quality and gene expression of porcine blastocysts after supplementations of in vitro culture medium and/or vitrification-warming media. Embryo quality, in terms of total cell number (TCN), DNA fragmentation and peroxide levels, together with the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), POU class 5 homeobox 1 (POU5F1) and heat shock protein 70 (HSPA1A), was analysed. In Experiment 1, gene expression and embryo quality of fresh blastocysts were evaluated after culture with or without L-ascorbic acid; no significant differences were observed between the groups. In Experiment 2, blastocysts cultured with or without L-ascorbic acid were vitrified using two different vitrification solutions, supplemented or not with L-ascorbic acid. Supplementation of culture and vitrification media significantly enhanced survival rates and reduced peroxide levels. No significant differences in TCN, DNA fragmentation and BAX, BCL2L1 and POU5F1 expression were found in vitrified blastocysts among experimental groups. Vitrification procedures increase HSPA1A transcript abundance, but this increase was significantly lower in embryos cultured and/or vitrified with L-ascorbic acid. Thus, supplementing culture and/or vitrification media with L-ascorbic acid enhances survival rates of porcine blastocysts, suggesting a relationship with HSPA1A expression.

Impact of light irradiation on preservation and function of mammalian spermatozoa

Light irradiation has been demonstrated to exert positive effects on gametes, and particularly on sperm. In effect, a high number of studies conducted in several species, including humans, mice, pigs, cattle and sheep, and using different light sources (such as lasers and light-emitting diodes) have demonstrated that photo-stimulation increases sperm motility. In addition, other works have shown that sperm fertilizing ability both in vitro and in vivo can be increased following light irradiation; there are also some evidences pointing out to an extend of lifespan of preserved semen. Notwithstanding, no study has reported a detrimental effect of visible light on DNA integrity. The mechanisms through which light exerts its effects are not completely elucidated, but mounting evidence gives cell photosensitizers, especially those present in the mitochondria, a vital role. Stimulating these molecules turns into an increase in the production of ATP and Ca2+ influx, which contributes to explain the effects of light upon spermatozoa. Additionally, the presence of opsins in spermatozoa as well as the potential influence of light on the conformation of other proteins may also be involved in the sperm response to light. However, there are still a significant number of points that need to be addressed and their elucidation may contribute to increase the utilization of light irradiation for sperm preservation and ART.

Melatonin affects the motility and adhesiveness of in vitro capacitated boar spermatozoa via a mechanism that does not depend on intracellular ROS levels

This work sought to address the effects of melatonin during in vitro capacitation (IVC) and progesterone‐induced acrosome exocytosis (IVAE) in boar spermatozoa. With this purpose, two different experiments were set. In the first one, IVC and IVAE were induced in the absence or presence of melatonin, which was added either at the start of IVC or upon triggering the IVAE with progesterone. Different parameters were evaluated, including intracellular levels of peroxides and superoxides, free cysteine radicals and distribution of specific lectins. While melatonin neither affected most capacitation‐associated parameters nor IVAE, it dramatically decreased sperm motility, with a maximal effect at 5 μm. This effect was accompanied by a significant increase in the percentage of agglutinated spermatozoa, which was independent from noticeable changes in the distribution of lectins. Levels of free cysteine radicals were significantly lower in melatonin treatments than in the control after 4 h of incubation in capacitating medium. The second experiment evaluated the effects of melatonin on in vitro fertilising ability of boar spermatozoa. Spermatozoa previously subjected to IVC in the presence of 1 μm melatonin and used for in vitro fertilisation exhibited less ability to bind the zona pellucida (ZP) and higher percentages of monospermy. In conclusion, melatonin affects sperm motility and the stability of nucleoprotein structure and also modulates the ability of in vitro capacitated boar spermatozoa to bind the oocyte ZP. However, such effects do not seem to be related to either its antioxidant properties or changes in the sperm glycocalix.