Papers by alfonso gutierrez-adan
Development to the blastocyst stage, the oxidative state, and the quality of early developmental ... more Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions
International Journal of Molecular Sciences, Jun 9, 2020
Minor splicing plays an important role in vertebrate development. Zrsr1 and Zrsr2 paralog genes h... more Minor splicing plays an important role in vertebrate development. Zrsr1 and Zrsr2 paralog genes have essential roles in alternative splicing, mainly participating in the recognition of minor (U12) introns. To further explore their roles during early embryo development, we produced Zrsr1 mu and Zrsr2 mu mutant mice, containing truncating mutations within the second zinc finger domain. Both homozygous mutant mice were viable with a normal lifespan. When we crossed a homozygous Zrsr2 mu/mu female with Zrsr1 mu/mu male, the double heterozygotes were non-viable, giving rise to embryos that stopped developing mainly between the 2-and 4-cell stages, just after zygotic gene activation. RNA-seq analysis of Zrsr1/2 mu 2-cell embryos showed altered gene and isoform expression of thousands of genes enriched in gene ontology terms and biological pathways related to ribosome, RNA transport, spliceosome, and essential zygotic gene activation steps. Alternative splicing was analyzed, showing a significant increase in intron retention in both U2 and U12 intron-containing genes related to cell cycle and mitotic nuclear division. Remarkably, both Zrsr1 and Zrsr2 were required for the conversion of mouse-induced pluripotent stem cells into 2C-like cells. According to our results, Zrsr1 or Zrsr2 are necessary for ZGA and both are indispensable for the conversion of induced pluripotent stem cells into 2C-like cells.
Archivos De Medicina Veterinaria, 2009
Transgenic biotechnology is a powerful tool for the generation of genetically modified animals wi... more Transgenic biotechnology is a powerful tool for the generation of genetically modified animals with applications in various fields such as veterinary, agriculture and biomedicine. Sperm mediated gene transfer (SMGT) is an interesting tool for animal transgenesis consisting on the intrinsic ability of the spermatic cells to bind and internalize exogenous DNA and allow their transfer into oocytes after fertilization, to become part of the genome of the new embryo. The seminal plasma plays an important role acting as a natural barrier and protecting the spermatozoa from exogenous molecules that could compromise their integrity. So, the epididymal spermatozoa are a valuable model to explore the possible effect of seminal plasma components. The objective of this study was to evaluate the interaction among sperm and transgene using Epididymal (EP) vs Ejaculated (EJ) sperm without seminal plasma. Linealized plasmid (GFP) (5.7 kb) labelled with fluorescein was added (1x10 8 spermatozoa/ml + 5µg DNA/ml) and incubated at 16 ºC. DNA binding and viability were measured simultaneously by flow cytometry during 120 minutes of incubation. The results showed that EP spermatozoa present a similar DNA-binding ability (12.63 ± 1.23% vs 10.94 ± 1.05%, P = 0.31) and viability throughout the incubation (14.64 ± 0.94% vs 13.42 ± 0.61%, P = 0.23) than EJ. We only detected a greater percentage of living DNA-bound spermatozoa in EP compared to EJ (2.10 ± 0.33% vs 1.05 ± 0.14%, P < 0.01). The DNA-binding was associated mainly to dead sperm or with low viability in both groups (EP: 10.53 ± 1.01% vs EJ: 9.89 ± 0.97%, P = 0.98). These results open new ways to explore and use epididymal spermatozoa in diverse applications (artificial insemination, in vitro fertilization and ICSI) associated with SMGT method.
Reproduction, May 1, 2011
In adult tissues, sexual dimorphism is largely attributed to sex hormone effects, although there ... more In adult tissues, sexual dimorphism is largely attributed to sex hormone effects, although there is increasing evidence for a major role of sex chromosome dosage. During preimplantation development, male and female embryos can display phenotypic differences that can only be attributed to the transcriptional differences resulting from their different sex chromosome complements. Thus, all expressed Y-linked genes and those X-linked genes that totally or partially escape X-chromosome inactivation at each specific developmental stage display transcriptional sexual dimorphism. Furthermore, these differentially expressed sex chromosome transcripts can regulate the transcription of autosomal genes, leading to a large transcriptional sexual dimorphism. The sex-dependent transcriptional differences may affect several molecular pathways such as glucose metabolism, DNA methylation and epigenetic regulation, and protein metabolism. These molecular differences may have developmental consequences, including sex-selective embryo loss and sex-specific epigenetic responses to environmental hazards, leading to long-term effects. This review discusses transcriptional sexual dimorphism in preimplantation embryos, its consequences on sex ratio biases and on the developmental origin of health and disease, and its significance for transcriptional studies and adult sexual dimorphism.
Journal of animal science and biotechnology, Jan 11, 2023
Background Sperm migration by thermotaxis is a guidance mechanism that operates along the oviduct... more Background Sperm migration by thermotaxis is a guidance mechanism that operates along the oviduct and it has proved to be a valid method for selecting spermatozoa with low DNA fragmentation (SDF) in mice, humans, and stallions. This study aimed to analyse if bull spermatozoa could be selected by thermotaxis and to assess their quality in terms of SDF as well as determine the presence of a specific sperm subpopulation based on sperm morphometry and assess their fertilizing capacity by ICSI. We used frozen-thawed sperm from 6 bulls and sperm selection by thermotaxis was performed with TALP medium supplemented with 25 mmol/L of HEPES and 5 mmol/L of caffeine. In these conditions, sperm selection was achieved, obtaining a net thermotaxis of 3.6%. Subsequently, we analysed the SDF of the migrated and notmigrated spermatozoa using the neutral COMET assay, and we evaluated the size of the sperm head using Hemacolor ® staining with Motic Images Plus 3 software. Additionally, migrated and not-migrated spermatozoa by thermotaxis were used to fertilize bovine in vitro matured (IVM) oocytes by ICSI, a very inefficient procedure in cattle that is only successful when the oocyte is artificially activated. The results showed lower SDF (χ², P < 0.001, 13.3% reduction, n = 8) and lower head size parameters (length and width, P < 0.01; and perimeter and area, P < 0.001; n = 4) in those spermatozoa migrated in comparison to those not-migrated. The distribution of sperm subpopulations structure varied between groups, highlighting cluster 2, characterized by spermatozoa with small head size, and high ellipticity and elongated heads, as the most abundant in the thermotaxis migrated group. When performed ICSI (without oocyte artificial activation) with the thermotactic sperm, the blastocyst rate was 32.2% ± 9.3% in the group microinjected with the thermotactic spermatozoa vs. 8.3% ± 7.8% in the group of not-migrated sperm (χ², P < 0.05). Our results showed that bull sperm selection by thermotaxis has a much higher DNA integrity, small and elongated head size parameters, and different sperm subpopulation structure than the not-selected spermatozoa. Additionally, we evidenced that thermotactic spermatozoa improve ICSI success rates.
Una alternativa biotecnológica para la producción de animales transgénicos es la transgénesis med... more Una alternativa biotecnológica para la producción de animales transgénicos es la transgénesis mediada por espermatozoides (SMGT), basada en la habilidad de las células espermáticas de unir e interiorizar ADN exógeno. En este estudio se plantearon dos objetivos principales: 1) evaluar el efecto de la presencia de ADN exógeno sobre la calidad seminal y 2) evaluar la eficiencia en la producción de embriones transgénicos porcinos in vivo mediante inseminación intrauterina quirúrgica con espermatozoides incubados con el transgén. Para alcanzar estos objetivos, los espermatozoides (libres de plasma seminal) fueron incubados en presencia del transgén EGFP (proteína verde fluorescente) durante 2 h a 16°C (en una relación de 10 8 cels/mL y 5 µg ADN/mL). Se valoró la motilidad, motilidad progresiva e integridad de membrana de los espermatozoides incubados, en presencia o ausencia del transgén. Los resultados mostraron que la presencia del ADN no afectó a ninguno de los parámetros seminales estudiados. Con muestras seminales incubadas con el transgén se llevaron a cabo inseminaciones intrauterinas mediante laparotomía en 4 hembras prepúberes. A los 6-7 días tras la inseminación se recolectaron los embriones, para evaluar la tasa de fecundación y la expresión de la proteína verde fluorescente EGFP en los mismos. El número medio de cuerpos lúteos por cerda fue de 10,50 ± 2,90 con una tasa de recolección de 11,90%. Se recolectó un total de 5 embriones presentando todos ellos un estado normal de desarrollo y una alta cali-dad (dos de ellos presentaban más de 400 células por embrión). Cuando fue analizada la expresión mediante microscopia de fluorescencia, ninguno de ellos expresaba la proteína EGFP. Este resultado, bajo las condiciones experimentales del presente estudio, podría indicar que el transgén se una en su mayoría a espermatozoides con baja viabilidad por lo que la probabilidad de que los espermatozoides vivos unan el ADN y sean capaces de fecundar en comparación con espermatozoides sin el transgén es realmente baja.
Reproduction, 2016
Offspring telomere length (TL) has been correlated with paternal TL, but the mechanism for this p... more Offspring telomere length (TL) has been correlated with paternal TL, but the mechanism for this parent of origin-specific inheritance remains unclear. The objective of this study has been to determine the role of spermatozoa TL in embryonic telomere lengthening by using two mouse models showing dimorphism in their spermatozoa TL: Mus musculus vs Mus spretus and old vs young Mus musculus. Mus spretus spermatozoa displayed a shorter TL than Mus musculus. Hybrid offspring exhibited lower TL compared with Mus musculus starting at the two-cell stage, before the onset of telomerase expression. To analyze the role of spermatozoa telomeres in early telomere lengthening, we compared the TL in oocytes, zygotes, two-cell embryos and blastocysts produced by parthenogenesis or by fertilization with Mus musculus or Mus spretus spermatozoa. TL was significantly higher in spermatozoa compared with oocytes, and it increased significantly from the oocyte to the zygote stage in those embryos fertilized with Mus musculus spermatozoa, but not in those fertilized with Mus spretus spermatozoa or produced by parthenogenesis. A further increase was noted from the zygote to the two-cell stage in fertilized Mus musculus embryos, whereas hybrid embryos maintained the oocyte TL. Spermatozoa TL shortened with age in Mus musculus and the offspring from young males showed a significantly higher TL compared with that fathered by old males. These significant differences were already noticeable at the two-cell stage. These results suggest that spermatozoa telomeres act as a guide for telomerase-independent telomere lengthening resulting in differences in TL that persist after birth.
Proceedings of the National Academy of Sciences of the United States of America, Feb 23, 2010
Data deposition: Raw data from microarray experiments was submitted to the Gene Expression Omnibu... more Data deposition: Raw data from microarray experiments was submitted to the Gene Expression Omnibus database (). The platform ID is GPL2112. The accession ID for the set of experiments described is GSE17921. A new 1382 pb cDNA was identified YZRSR2 (GQ426330).
Reproduction, Jun 1, 2011
Sex chromosome transcripts can lead to a broad transcriptional sexual dimorphism in the absence o... more Sex chromosome transcripts can lead to a broad transcriptional sexual dimorphism in the absence of concomitant or previous exposure to sex hormones, especially when X-chromosome inactivation (XCI) is not complete. XCI timing has been suggested to differ greatly among species, and in bovine, most of the X-linked transcripts are upregulated in female blastocysts. To determine the timing of XCI, we analyzed in day 14 bovine embryos the sexual dimorphic transcription of seven X-linked genes known to be upregulated in female blastocysts (X24112, brain-expressed X-linked 2 (BEX2), ubiquitin-conjugating enzyme E2A (UBE2A), glucose-6-phosphate dehydrogenase (G6PD), brain-expressed X-linked 1 (BEX1), calpain 6 (CAPN6), and spermidine/spermine N-acetyltransferase 1 (SAT1)). The transcription of five genes whose expression differs between sexes at the blastocyst stage (DNMT3A, interferon tau (IFNT2), glutathione S-transferase mu 3 (GSTM3), progesterone receptor membrane component 1 (PGRMC1), and laminin alpha 1 (LAMA1)) and four genes related with sex determination (Wilms tumor 1 (WT1), gata binding protein 4 (GATA4), zinc finger protein multitype 2 (ZFPM2), and DMRT1) was also analyzed to determine the evolution of transcriptional sexual dimorphism. The expression level of five X-linked transcripts was effectively equalized among sexes suggesting that, in cattle, a substantial XCI occurs during the period between blastocyst hatching and initiation of elongation, although UBE2A and SAT1 displayed significant transcriptional differences. Similarly, sexual dimorphism was also reduced for autosomal genes with only DNMT3A and IFNT2 exhibiting sex-related differences. Among the genes potentially involved in sex determination, Wilms tumor 1 (WT1) was significantly upregulated in males and GATA4 in females, whereas no differences were observed for ZFPM2 and DMRT1. In conclusion, a major XCI occurred between the blastocyst and early elongation stages leading to a reduction in the transcriptional sexual dimorphism of autosomal genes, which makes the period the most susceptible to sex-specific embryo loss.
Scientific Reports, Jun 3, 2021
In vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to ... more In vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (C DMSO ) during minor (2 to 8-cell stage; MN EGA ) or major (8 to 16-cell stage; MJ EGA ) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MN EGA phase and in Nob10 (61.0 ± 0.8%) for MJ EGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.
Reproduction, Fertility and Development, 2004
The aim of the current study was to identify the bovine homolog of the murine Ped (preimplantatio... more The aim of the current study was to identify the bovine homolog of the murine Ped (preimplantation embryo development) gene, which regulates mouse preimplantation embryonic growth, including cleavage rate and embryo survivability, and to characterize the expression pattern of this gene during bovine preimplantation embryo development. Experiment (I): The National Center for Biotechnology Information (NCBI) GenBank/EBI EST databases were searched for bovine-expressed sequence tags (EST) that were homologous with the murine Ped gene (Accession number: NM_010394). The resulting ESTs were aligned and assembled in to one complete sequence (841bp), which was shown to be homologous with the Murine Ped gene and the Bovine Major Histocompatibility Complex class I 4221.1 gene (Accession No.: AJ010865, length 1090bp). The expression of the protein product of the Ped gene by bovine tissue was confirmed using Western Blot analysis. Experiment (II): The expression pattern of the Ped gene homolog ...
Reproduction, Fertility and Development, 2005
There is not a simple system that allows us to identify stem cells in adult tissues. Cells of adu... more There is not a simple system that allows us to identify stem cells in adult tissues. Cells of adult tissues arise from dividing progenitor cells, which themselves are derived from multipotential stem cells. Telomerase is the enzyme that maintains the ends of linear chromosomes in eukaryotic cells. Recently, a segment of the promoter sequence of the reverse transcriptase of murine telomerase (mTert) has been characterized. mTert is expressed with greatest abundance during embryogenesis and becomes widely expressed in adult tissues at low levels. This low expression level in adult tissues may be due to the presence of pluripotent stem cells present in those tissues. To examine the relationship between telomerase activity and multipotential of adult cells we have generated three constructs (1k-, 2k-, and 5k-mTert-GFP) comprising different segments of the mTert promoter sequence coupled to the coding sequence of the green fluorescent protein (GFP). These constructs were electroporated i...
Human Reproduction, 2016
study question: Does a novel antioxidant formulation designed to restore redox balance within the... more study question: Does a novel antioxidant formulation designed to restore redox balance within the male reproductive tract, reduce sperm DNA damage and increase pregnancy rates in mouse models of sperm oxidative stress? summary answer: Oral administration of a novel antioxidant formulation significantly reduced sperm DNA damage in glutathione peroxidase 5 (GPX5), knockout mice and restored pregnancy rates to near-normal levels in mice subjected to scrotal heat stress. what is known already: Animal and human studies have documented the adverse effect of sperm DNA damage on fertilization rates, embryo quality, miscarriage rates and the transfer of de novo mutations to offspring. Semen samples of infertile men are known to be deficient in several key antioxidants relative to their fertile counterparts. Antioxidants alone or in combination have demonstrated limited efficacy against sperm oxidative stress and DNA damage in numerous human clinical trials, however these studies have not been definitive and an optimum combination has remained elusive. study design, size, duration: The efficacy of the antioxidant formulation was evaluated in two well-established mouse models of oxidative stress, scrotal heating and Gpx5 knockout (KO) mice, (n ¼ 12 per experimental group), by two independent laboratories. Mice were provided the antioxidant product in their drinking water for 2 -8 weeks and compared with control groups for sperm DNA damage and pregnancy rates. participants/materials, setting, methods: In the Gpx5 KO model, oxidative DNA damage was monitored in spermatozoa by immunocytochemical detection of 8-hydroxy-2 ′ -deoxyguanosine (8OHdG). In the scrotal heat stress model, male fertility was tested by partnering with three females for 5 days. The percentage of pregnant females, number of vaginal plugs, resorptions per litter, and litter size were recorded. main results and role of chance: Using immunocytochemical detection of 8OHdG as a biomarker of DNA oxidation, analysis of control mice revealed that around 30% of the sperm population was positively stained. This level increased to about 60% in transgenic mice deficient in the antioxidant enzyme, GPX5. Our results indicate that an 8 week pretreatment of Gpx5 KO mice with the antioxidant formulation provided complete protection of sperm DNA against oxidative damage. In mouse models of scrotal heat stress, only 35% (19/54) of female mice became pregnant resulting in 169 fetuses with 18% fetal resorption (30/169). This is in contrast to the antioxidant pretreated group where 74% (42/57) of female mice became pregnant, resulting in 427 fetuses with 9% fetal resorption (38/427). In both animal models the protection provided by the novel antioxidant was statistically significant (P , 0.01 for the reduction of 8OHdG in the spermatozoa of Gpx5 KO mice and P , 0.05 for increase in fertility in the scrotal heat stress model).
BMC Genomics, 2016
Background: Since the development of in vitro embryo production in cattle, different supplements ... more Background: Since the development of in vitro embryo production in cattle, different supplements have been added to culture media to support embryo development, with serum being the most popular. However, the addition of serum during embryo culture can induce high birthweights and low viability in calves (Large Offspring Syndrome). Analysis of global gene expression in bovine embryos produced under different conditions can provide valuable information to optimize culture media for in vitro embryo production. Results: We used RNA sequencing to examine the effect of in vitro embryo production, in either serum-containing or serum-free media, on the global gene expression pattern of individual bovine blastocysts. Compared to in vivo derived embryos, embryos produced in serum-containing medium had five times more differentially expressed genes than embryos produced in serum-free conditions (1109 vs. 207). Importantly, in vitro production in the presence of serum appeared to have a different impact on the embryos according to their sex, with male embryos having three times more genes differentially expressed than their female counterparts (1283 vs. 456). On the contrary, male and female embryos produced in serum-free conditions showed the same number (191 vs. 192) of genes expressed differentially; however, only 44 of those genes were common in both comparisons. The pathways affected by in vitro production differed depending on the type of supplementation. For example, embryos produced in serum-containing conditions had a lower expression of genes related to metabolism while embryos produced in serum-free conditions showed aberrations in genes involved in lipid metabolism. Conclusions: Serum supplementation had a major impact on the gene expression pattern of embryos, with male embryos being the most affected. The transcriptome of embryos produced in serum-free conditions showed a greater resemblance to that of in vivo derived embryos, although genes involved in lipid metabolism were altered. Male embryos appeared to be most affected by suboptimal in vitro culture, i.e. in the presence of serum.
Reproduction, Fertility and Development, 2012
Reduced oocyte and embryo quality are recognised as major factors in the problem of disappointing... more Reduced oocyte and embryo quality are recognised as major factors in the problem of disappointing fertility in high producing dairy cows. This review aims to shed more light on the importance of the intrafollicular environment in the subfertility problem in dairy cows. Metabolic disturbances associated with negative energy balance (NEB) early postpartum are associated with ovarian dysfunction. Changes in the growth pattern of the ovarian follicle during a period of NEB can indirectly affect oocyte quality. Furthermore, a maternal metabolic disorder (linked with NEB or nutritionally induced) may alter the endocrine and biochemical composition of the follicular fluid, the micro-environment of the growing and maturing female gamete. The maturing oocyte is very sensitive to any perturbation in its direct environment and in vitro maturation models revealed that some of these metabolic changes reduce the oocyte’s developmental competence. Also, embryo quality is significantly reduced due ...
Archivos De Medicina Veterinaria, 2009
Transgenic biotechnology is a powerful tool for the generation of genetically modified animals wi... more Transgenic biotechnology is a powerful tool for the generation of genetically modified animals with applications in various fields such as veterinary, agriculture and biomedicine. Sperm mediated gene transfer (SMGT) is an interesting tool for animal transgenesis consisting on the intrinsic ability of the spermatic cells to bind and internalize exogenous DNA and allow their transfer into oocytes after fertilization, to become part of the genome of the new embryo. The seminal plasma plays an important role acting as a natural barrier and protecting the spermatozoa from exogenous molecules that could compromise their integrity. So, the epididymal spermatozoa are a valuable model to explore the possible effect of seminal plasma components. The objective of this study was to evaluate the interaction among sperm and transgene using Epididymal (EP) vs Ejaculated (EJ) sperm without seminal plasma. Linealized plasmid (GFP) (5.7 kb) labelled with fluorescein was added (1x10 8 spermatozoa/ml + 5µg DNA/ml) and incubated at 16 ºC. DNA binding and viability were measured simultaneously by flow cytometry during 120 minutes of incubation. The results showed that EP spermatozoa present a similar DNA-binding ability (12.63 ± 1.23% vs 10.94 ± 1.05%, P = 0.31) and viability throughout the incubation (14.64 ± 0.94% vs 13.42 ± 0.61%, P = 0.23) than EJ. We only detected a greater percentage of living DNA-bound spermatozoa in EP compared to EJ (2.10 ± 0.33% vs 1.05 ± 0.14%, P < 0.01). The DNA-binding was associated mainly to dead sperm or with low viability in both groups (EP: 10.53 ± 1.01% vs EJ: 9.89 ± 0.97%, P = 0.98). These results open new ways to explore and use epididymal spermatozoa in diverse applications (artificial insemination, in vitro fertilization and ICSI) associated with SMGT method.
Reproduction Nutrition Development, 1999
The present review has two goals. First, to offer an overview of recent advances in the technical... more The present review has two goals. First, to offer an overview of recent advances in the technical strategies applied to the production of transgenic large domestic animals, and second, to review how transgenic technology can be applied to the modification of milk composition. Transgenic sheep and cattle obtained through nuclear transfer are now a reality, opening up a means of ruminant transgenic production with an efficiency that entitles us to consider it a serious alternative to microin- jection. Nuclear transfer also consistently reduces the time needed to establish a transgenic production herd, and what is more important, it opens up the way to homologous recombination in large species, which at the moment is restricted to mice. Other interesting technological contributions have also taken place lately, some of them towards the modification of the male germ line, and oth- ers developing viral vectors with the ability to alter the genetic information of animals. The simpli- fication of the methodology and the consistent reduction of the time needed to carry out a trans- genic experiment will allow us to test several hypotheses directed at the modification of milk components. This may help towards the application of transgenic technology in the dairy industry, which unlike pharmaceutical companies, has been somehow reluctant over these approaches. © Inra/Elsevier, Paris transgenesis / milk / dairy industry / cattle Résumé ― Transgenèse chez les grands mammifères domestiques, développement à venir de
Sperm-Mediated Gene Transfer: Concepts and Controversies, 2012
Una alternativa biotecnológica para la producción de animales transgénicos es la transgénesis med... more Una alternativa biotecnológica para la producción de animales transgénicos es la transgénesis mediada por espermatozoides (SMGT), basada en la habilidad de las células espermáticas de unir e interiorizar ADN exógeno. En este estudio se plantearon dos objetivos principales: 1) evaluar el efecto de la presencia de ADN exógeno sobre la calidad seminal y 2) evaluar la eficiencia en la producción de embriones transgénicos porcinos in vivo mediante inseminación intrauterina quirúrgica con espermatozoides incubados con el transgén. Para alcanzar estos objetivos, los espermatozoides (libres de plasma seminal) fueron incubados en presencia del transgén EGFP (proteína verde fluorescente) durante 2 h a 16°C (en una relación de 10 8 cels/mL y 5 µg ADN/mL). Se valoró la motilidad, motilidad progresiva e integridad de membrana de los espermatozoides incubados, en presencia o ausencia del transgén. Los resultados mostraron que la presencia del ADN no afectó a ninguno de los parámetros seminales estudiados. Con muestras seminales incubadas con el transgén se llevaron a cabo inseminaciones intrauterinas mediante laparotomía en 4 hembras prepúberes. A los 6-7 días tras la inseminación se recolectaron los embriones, para evaluar la tasa de fecundación y la expresión de la proteína verde fluorescente EGFP en los mismos. El número medio de cuerpos lúteos por cerda fue de 10,50 ± 2,90 con una tasa de recolección de 11,90%. Se recolectó un total de 5 embriones presentando todos ellos un estado normal de desarrollo y una alta cali-dad (dos de ellos presentaban más de 400 células por embrión). Cuando fue analizada la expresión mediante microscopia de fluorescencia, ninguno de ellos expresaba la proteína EGFP. Este resultado, bajo las condiciones experimentales del presente estudio, podría indicar que el transgén se una en su mayoría a espermatozoides con baja viabilidad por lo que la probabilidad de que los espermatozoides vivos unan el ADN y sean capaces de fecundar en comparación con espermatozoides sin el transgén es realmente baja.
Theriogenology, 2009
Intracytoplasmic sperm injection-sperm-mediated gene transfer (ICSI-SMGT) is a useful tool for th... more Intracytoplasmic sperm injection-sperm-mediated gene transfer (ICSI-SMGT) is a useful tool for the production of transgenic mice but is still rather inefficient in farm animals. In the current study, we evaluated the effect of the sperm treatments on the efficiency for producing enhanced green fluorescent protein (EGFP)-expressing pig embryos by ICSI-SMGT. Four different sperm treatments were assayed: (1) fresh (control), (2) frozen-thawing (FT), (3) quick freezing without cryoprotectant agents (QF), and (4) Triton X-100 treatment (TX-100). First, we evaluated the DNA-binding ability and the viability of sperm under the different treatments coincubated with exogenous DNA (EGFP) by flow cytometry. Second, we evaluated the embryo production rate and the efficiency in transgene expression in embryos after using these spermatozoa to fertilize oocytes by ICSI. Sperm treatment significantly increased DNA-binding capacity but reduced sperm viability compared with that of the control group. Treatments damaging the spermatozoa's membranes (QF and TX-100) resulted in a greater capacity of sperm binding exogenous DNA than that after FT treatment (P < 0.01). Similar rates of EGFP-expressing embryos were obtained from the control, FT, and TX-100 groups (37.04 AE 3.52%, 43.54 AE 5.41%, and 29.03 AE 8.29%, respectively), but were significantly higher in the QF group (80.43 AE 5.91%). These results demonstrate that the integrity of the sperm plasma membrane plays a critical role in DNA interaction, and altered plasma membranes facilitate interactions between an injected exogenous DNA and the sperm chromatin. However, severe sperm treatments such as QF and TX-100 may damage the sperm nucleus, induce DNA fragmentation, and/or lead to chromosomal breakage with a detrimental effect on further embryonic development.
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Papers by alfonso gutierrez-adan