Papers by Karina Gutierrez
International Journal of Molecular Sciences
DNA damage in early-stage embryos impacts development and is a risk factor for segregation of alt... more DNA damage in early-stage embryos impacts development and is a risk factor for segregation of altered genomes. DNA damage response (DDR) encompasses a sophisticated network of proteins involved in sensing, signaling, and repairing damage. DDR is regulated by reversible post-translational modifications including acetylation, methylation, phosphorylation, ubiquitylation, and SUMOylation. While important regulators of these processes have been characterized in somatic cells, their roles in early-stage embryos remain broadly unknown. The objective of this study was to explore how ubiquitylation and SUMOylation are involved in the regulation of early development in porcine embryos by assessing the mRNA profile of genes encoding ubiquitination (UBs), deubiquitination (DUBs), SUMOylation (SUMOs) or deSUMOylation (deSUMOs) enzymes in oocyte and embryos at different stages of development, and to evaluate if the induction of DNA damage at different stages of embryo development would alter the...
There is growing evidence that histone lysine demethylases (KDMs) play critical roles in the regu... more There is growing evidence that histone lysine demethylases (KDMs) play critical roles in the regulation of embryo development. This study investigated if KDM7A, a lysine demethylase known to act on mono-(me1) and di-(me2) methylation of H3K9 and H3K27, participates in the regulation of early embryo development. Knockdown of <i>KDM7A</i> mRNA reduced blastocyst formation by 69.2% in <i>in vitro</i> fertilized (IVF), 48.4% in parthenogenetically activated (PA), and 48.1% in somatic cell nuclear transfer (SCNT) embryos compared to controls. Global immunofluorescence (IF) signal in <i>KDM7A</i> knockdown compared to control embryos was increased for H3K27me1 on D7, for H3K27me2 on D3 and D5, for H3K9me1 on D5 and D7, and for H3K9me2 on D5 embryos, but decreased for H3K9me1, me2 and me3 on D3. Moreover, <i>KDM7A</i> knockdown altered mRNA expression, including the downregulation of <i>KDM3C</i> on D3, <i>NANOG</i> on...
Life, 2022
CRISPR/Cas9 technology is a powerful tool used for genome manipulation in different cell types an... more CRISPR/Cas9 technology is a powerful tool used for genome manipulation in different cell types and species. However, as with all new technologies, it still requires improvements. Different factors can affect CRISPR/Cas efficiency in zygotes, which influence the total cost and complexity for creating large-animal models for research. This study evaluated the importance of zygote cell cycle stage between early-injection (within 6 h post activation/fertilization) versus late-injection (14–16 h post activation/fertilization) when the CRISPR/Cas9 components were injected and the inhibition of the homologous recombination (HR) pathway of DNA repair on gene editing, embryo survival and development on embryos produced by fertilization, sperm injection, somatic cell nuclear transfer, and parthenogenetic activation technologies. Injections at the late cell cycle stage decreased embryo survival (measured as the proportion of unlysed embryos) and blastocyst formation (68.2%; 19.3%) compared to ...
Biology of Reproduction, 2021
Conditions of impaired energy and nutrient homeostasis, such as diabetes and obesity, are associa... more Conditions of impaired energy and nutrient homeostasis, such as diabetes and obesity, are associated with infertility. Hyperglycemia increases endoplasmic reticulum stress as well as oxidative stress and reduces embryo development and quality. Oxidative stress also causes deoxyribonucleic acid damage, which impairs embryo quality and development. The natural bile acid tauroursodeoxycholic acid reduces endoplasmic reticulum stress and rescues developmentally incompetent late-cleaving embryos, as well as embryos subjected to nuclear stress, suggesting the endoplasmic reticulum stress response, or unfolded protein response, and the genome damage response are linked. Tauroursodeoxycholic acid acts via the Takeda-G-protein-receptor-5 to alleviate nuclear stress in embryos. To evaluate the role of tauroursodeoxycholic acid/Takeda-G-protein-receptor-5 signaling in embryo unfolded protein response, we used a model of glucose-induced endoplasmic reticulum stress. Embryo development was impai...
Animal reproduction, 2015
Trimethylation of histone H3 on lysine 27 (H3K27me3) is established by Polycomb Repressive Comple... more Trimethylation of histone H3 on lysine 27 (H3K27me3) is established by Polycomb Repressive Complex 2 (PRC2) and it is associated with stable and heritable gene silencing. In pluripotent cells, genes associated with development and cell differentiation are maintained repressed by H3K27me3. However, this process is not fully understood. The AdoHcy hydrolase inhibitor 3-Deazaneplanocin A (DZNep) can block the action of the PRC2enzymes and thereby inhibit H3K27me3. In this study, we evaluated the effect of treating bovine embryos during in vitro development with DZNep on the expression of genes encoding PRC2 enzymes (EZH2, EED and SUZ12), and transcription factors regulating cell pluripotency (OCT4 and NANOG) and trophoblast differentiation (CDX2). Oocytes obtained from slaughterhouse ovaries were subjected to in vitro maturation (IVM) for 24 h at 38.5°C, with 5% CO2 in air and saturated humidity. In vitro fertilization (IVF) was performed with a previously tested frozen-thawed semen fr...
Molecular Reproduction and Development, 2019
DNA damage associated with assisted reproductive technologies is an important factor affecting ga... more DNA damage associated with assisted reproductive technologies is an important factor affecting gamete fertility and embryo development. Activation of the TGR5 receptor by tauroursodeoxycholic acid (TUDCA) has been shown to reduce endoplasmic reticulum (ER) stress in embryos; however, its effect on genome damage responses (GDR) activation to facilitate DNA damage repair has not been examined. This study aimed to investigate the effect of TUDCA on DNA damage repair and embryo development. In a porcine model of ultraviolet light (UV)-induced nuclear stress, TUDCA reduced DNA damage and ER stress in developing embryos, as measured by γH2AX and glucose-regulated protein 78 immunofluorescence, respectively. TUDCA was equally able to rescue early embryo development. No difference in total cell number, DNA damage, or percentage of apoptotic cells, measured by cleaved caspase 3 immunofluorescence, was noted in embryos that reached the blastocyst stage. Interestingly, Dicer-substrate short interfering RNAmediated disruption of TGR5 signaling abrogated the beneficial effects of TUDCA on UV-treated embryos. Quantitative PCR analysis revealed activation of the GDR, through increased messenger RNA abundance of DNAPK, 53BP1, and DNA ligase IV, as well as the ER stress response, through increased spliced XBP1 and X-linked inhibitor of apoptosis. Results from this study demonstrated that TUDCA activates TGR5-mediated signaling to reduce DNA damage and improve embryo development after UV exposure.
Animal Reproduction Science, 2021
There is growing evidence that greater than homeostatic blood concentrations of nonesterified fat... more There is growing evidence that greater than homeostatic blood concentrations of nonesterified fatty acids (NEFAs) and β-hydroxybutyrate (BHBA) have negative consequences on dairy cow's fertility, but effects on cell homeostasis in the reproductive system is not completely understood. In this study, lipids accumulation, reactive oxygen species (ROS) concentrations, abundance of gene transcripts, and immunofluorescence signal of H3K4me3 and H3K9me3 were evaluated in endometrial epithelial cells of cattle cultured with NEFAs (Oleic (OA), Stearic (SA) and Palmitic (PA) acids), BHBA, NEFAs + BHBA or each of the three NEFAs alone. The cellular lipids were in greater concentrations as a result of NEFAs + BHBA, NEFAs, SA or OA supplementation, but not by BHBA or PA. The ROS concentrations were greater when there were treatments with NEFAs + BHBA, NEFAs or BHBA. The relative mRNA abundance for genes involved in the regulation of apoptosis (XIAP), glucose transport (GLUT3), and DNA methylation (DNMT1) were greater when there were NEFAs + BHBA, but not NEFAs, BHBA, OA, SA or PA treatments. The immunofluorescence signal for H3K9me3 was greater when there were NEFAs + BHBA, NEFAs or PA, but not by BHBA, OA or SA treatments. These findings indicate that NEFAs and BHBA have an additive effect on endometrial cells of cattle by altering epigenetic markers and the expression of genes controlling important cellular pathways. Furthermore, there was cellular lipid accumulation and increased H3K9me3 in cultured bovine endometrial cells that was mainly induced by OA and PA treatments, respectively.
Frontiers in Cell and Developmental Biology, 2020
The lysine demethylases KDM5B and KDM5C are highly, but transiently, expressed in porcine embryos... more The lysine demethylases KDM5B and KDM5C are highly, but transiently, expressed in porcine embryos around the genome activation stage. Attenuation of KDM5B and KDM5C mRNA hampered embryo development to the blastocyst stage in fertilized, parthenogenetically activated and nuclear transfer embryos. While KDM5B attenuation increased H3K4me2-3 levels on D3 embryos and H3K4me1-2-3 on D5 embryos, KDM5C attenuation increased H3K9me1 on D3 embryos, and H3K9me1 and H3K4me1 on D5 embryos. The relative mRNA abundance of EIF1AX and EIF2A on D3 embryos, and the proportion of D4 embryos presenting a fluorescent signal for uridine incorporation were severely reduced in both KDM5B-and KDM5Cattenuated compared to control embryos, which indicate a delay in the initiation of the embryo transcriptional activity. Moreover, KDM5B and KDM5C attenuation affected DNA damage response and increased DNA double-strand breaks (DSBs), and decreased development of UV-irradiated embryos. Findings from this study revealed that both KDM5B and KDM5C are important regulators of early development in porcine embryos as their attenuation altered H3K4 and H3K9 methylation patterns, perturbed embryo genome activation, and decreased DNA damage repair capacity.
Epigenetics, 2019
The histone lysine demethylase KDM7A is required for normal development and first cell lineage sp... more The histone lysine demethylase KDM7A is required for normal development and first cell lineage specification in porcine embryos
Reproduction, 2018
Insufficient epigenetic reprogramming is incompatible with normal development of embryos produced... more Insufficient epigenetic reprogramming is incompatible with normal development of embryos produced by somatic cell nuclear transfer (SCNT), but treatment with histone deacetylases inhibitors (HDACi) enhances development of SCNT embryos. However, the mechanisms underpinning HDACi benefits in SCNT embryos remain largely uncharacterized. We hypothesized that, in addition to enhancing reprogramming, HDACi treatment may promote expression of genes not required for early development of SCNT embryos. To test this hypothesis, RNA synthesis was inhibited by treating bovine SCNT embryos with 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DBR), which were concomitantly treated or not with Scriptaid (Scrip; an HDACi). Development to the blastocyst stage was significantly increased by treatment with Scrip alone (26.6%) or associated with DRB (28.6%) compared to Control (17.9%). The total number of nuclei was significantly improved only in embryos that were treated with both Scrip + DRB. Nuclear ...
Theriogenology, 2019
Oocyte activation is physiologically triggered by the sperm during fertilization, however, produc... more Oocyte activation is physiologically triggered by the sperm during fertilization, however, production of porcine embryos by somatic cell nuclear transfer (SCNT), intracytoplasmic sperm injection (ICSI) or parthenogenetic activation (PA) requires artificial oocyte activation. Although effective protocols for artificial oocyte activation have been developed, current protocols require long exposures to non-specific inhibitors, which do not mimic the physiological process and may have detrimental consequences for embryo development. This study attempted to mimic the physiological activation events induced by fertilization, through the manipulation of Ca and Zn levels, and protein kinase C (PKC) as well as cyclin dependent kinase 1 (CDK1) activities, with the aim of developing an improved protocol for activation of porcine oocytes. In the first experiment, matured oocytes were exposed to ionomycin (Ion) for 5 min, and then treated with a specific CDK1 inhibitor (RO-3306) and/or PKC activ...
Molecular reproduction and development, Jan 9, 2018
Oocytes collected from prepubertal animals are known to be less developmentally competent than th... more Oocytes collected from prepubertal animals are known to be less developmentally competent than those from adult animals. There is evidence suggesting that acquisition of developmental competence in bovine oocytes may be linked to the expression profile of genes in the granulosa cells (GCs). Cumulus-oocyte complexes (COC) and GCs were collected from 12 Holstein heifers between 2 and 6 months of age (nine follicle-stimulating hormone [FSH] treated and three untreated) and eight FSH-treated cows. The COCs from prepubertal animals were matured, fertilized, and cultured in vitro to assess development to the blastocyst stage. The relative messenger RNA (mRNA) abundance of FSHR, StAR, CYP19A1, HSD3B1, CX43, FOXO1, and XIAP in GCs were quantified by real-time quantitative polymerase chain reaction. Results from this study revealed that GCs of prepubertal animals respond to FSH treatment by increasing mRNA levels of genes promoting estradiol synthesis and follicular growth ( FSHR and CYP19A1...
Theriogenology, Jan 17, 2018
Laparoscopic Ovum Pick-Up (LOPU) in calves followed by in vitro embryo production (IVEP) and tran... more Laparoscopic Ovum Pick-Up (LOPU) in calves followed by in vitro embryo production (IVEP) and transfer (ET) into adult recipients has great potential for accelerated genetic gain through shortening of the generation interval. In this study, 11 Holstein calves were subjected to up to six LOPU procedures between the ages of 2-6 months at 2-3 weeks interval. In all cases, the animals received a CIDR 5 days prior to LOPU and were gonadotropin-stimulated starting at 72 h before LOPU using one of three protocols that were rotated twice among the animals during the study. Calves were injected with FSH every 12 h (FSH12h), or every 8 h (FSH8h) or every 8 h until -36 h from LOPU at which point the FSH was replaced with a single dose of 400 IU eCG (FSH8h-eCG). No statistical differences were observed among the 3 treatments in terms of mean follicles available for aspiration (35.7 ± 16 vs. 38.5 ± 25 vs. 31.1 ± 22), mean oocytes recovered (26.5 ± 14 vs. 21.6 ± 10 vs. 19.4 ± 14) and cleavage rate...
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 21, 2017
DNA double-strand breaks (DSBs) are less frequent than single-strand breaks but have more harmful... more DNA double-strand breaks (DSBs) are less frequent than single-strand breaks but have more harmful consequences on cell survival and physiology. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are the two main pathways that are responsible for DSB repair in eukaryotic cells, but their importance for the preservation of genome stability in totipotent blastomeres of early developing embryos has not been determined. In this study, we observed that the chemical inhibition of HR or both pathways, but not NHEJ alone, increased the number of DSBs, reduced embryo development to the blastocyst stage, and resulted in embryos with higher proportions of apoptotic cells. Targeted knockdown of ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related; HR regulators) and DNA-dependent protein kinase (NHEJ regulator) mRNAs revealed that the attenuation of HR or both HR and NHEJ regulators severely impaired blastocyst formation and quality. Attenuation of ...
PloS one, 2017
Early-cleaving embryos are known to have better capacity to reach the blastocyst stage and produc... more Early-cleaving embryos are known to have better capacity to reach the blastocyst stage and produce better quality embryos compared to late-cleaving embryos. To investigate the significance of endoplasmic reticulum (ER) stress on early embryo cleavage kinetics and development, porcine embryos produced in vitro were separated into early- and late-cleaving groups and then cultured in the absence or presence of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Developing embryos were collected at days 3 to 7 of culture for assessment of ER stress status, incidence of DNA double-strand breaks (DSBs), development and total cell number. In the absence of TUDCA treatment, late-cleaving embryos exhibited ER stress, higher incidence of DNA DSBs, as well as reductions in development to the blastocyst stage and total embryo cell numbers. Treatment of late-cleaving embryos with TUDCA mitigated these effects and markedly improved embryo quality and development. These results demonstrate ...
Theriogenology, Jan 10, 2017
The use of oocytes recovered from prepubertal donors for in vitro embryo production has great pot... more The use of oocytes recovered from prepubertal donors for in vitro embryo production has great potential for accelerating the rate of genetic gain in the dairy industry. However, these oocytes are known to be less developmentally competent than those from adult donors. In this study, we investigated the effect of age and gonadotropin stimulation in Holstein heifers subjected to oocyte collection every two weeks between 2 and 6 months of age. In order to assess the effect of gonadotropin stimulation, animals were subjected to one of three treatments, namely Short (ST; 36-42 h), Long (LT; ≥72 h) and No Treatment (NT) prior to laparoscopic ovum pick-up (LOPU). Our results show that the LT significantly improved the proportion of large follicles (>5 mm diameter) present in the ovary (LT 34.0% vs. ST 11.2% vs. NT 2.4%, P < 0.05), as well as the percentage of good-quality cumulus oocyte complexes (COCs) recovered (LT 95.3 ± 18% vs. ST 85.4 ± 22% vs. NT 82.2 ± 14%, P < 0.05) and bl...
Peptides, 2016
The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cell... more The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cells highlights the possible effect of renin-independent prorenin in the resumption of meiosis in oocytes that was postulated in the 1980s.This study aimed to identify the (pro)renin receptor in the ovary and to assess the effect of prorenin on meiotic resumption. The (pro)renin receptor protein was detected in bovine cumulus-oocyte complexes, theca cells, granulosa cells, and in the corpus luteum. Abundant (pro)renin receptor messenger ribonucleic acid (mRNA) was detected in the oocytes and cumulus cells, while prorenin mRNA was identified in the cumulus cells only. Prorenin at concentrations of 10 −10 , 10 −9 , and 10 −8 M incubated with oocytes co-cultured with follicular hemisections for 15 h caused the resumption of oocyte meiosis. Aliskiren, which inhibits free renin and receptor-bound renin/prorenin, at concentrations of 10 −7 , 10 −5 , and 10 −3 M blocked this effect (P < 0.05). To determine the involvement of angiotensin II in prorenin-induced meiosis resumption, cumulus-oocyte complexes and follicular hemisections were treated with prorenin and with angiotensin II or saralasin (angiotensin II antagonist). Prorenin induced the resumption of meiosis independently of angiotensin II. Furthermore, cumulus-oocyte complexes cultured with forskolin (200 M) and treated with prorenin and aliskiren did not exhibit a prorenin-induced resumption of meiosis (P < 0.05). Only the oocytes' cyclic adenosine monophosphate levels seemed to be regulated by prorenin and/or forskolin treatment after incubation for 6 h. To the best of our knowledge, this is the first study to identify the (pro)renin receptor in ovarian cells and to demonstrate the independent role of prorenin in the resumption of oocyte meiosis in cattle.
Reproductive Toxicology, 2016
Busulfan is a chemotherapy drug that has side effects on spermatogonial stem cells (SSC). The eff... more Busulfan is a chemotherapy drug that has side effects on spermatogonial stem cells (SSC). The effects of bulsufan treatment on male germ cells and fertility vary significantly between individuals. In this study, we have used molecular, cellular and histopathology approaches to investigate the effects of a single intraperitoneal dose of busulfan (40mgkg(-1)) in two mice strains, Balb/C and Swiss, at two different periods after treatment, 30 and 90 days. Testicular degeneration was observed in both Balb/C and Swiss mice after busulfan injection. Interestingly, testicular functions and fertility recovered spontaneously post busulfan treatment in Swiss mice, but not in Balb/C mice. Abnormal fertility induced by busulfan in Balb/C mice was associated with altered seminiferous tubules, sperm morphology and transcript levels of Nanos2, Nanos3, Gdnf and Plzf genes. These findings revealed that SSC of Balb/C mice are more sensitive to the toxic effects of busulfan then those of Swiss mice.
Frontiers in Genetics, 2015
Since domestication, pigs have been used extensively in agriculture and kept as companion animals... more Since domestication, pigs have been used extensively in agriculture and kept as companion animals. More recently they have been used in biomedical research, given they share many physiological and anatomical similarities with humans. Recent technological advances in assisted reproduction, somatic cell cloning, stem cell culture, genome editing, and transgenesis now enable the creation of unique porcine models of human diseases. Here, we highlight the potential applications and advantages of using pigs, particularly minipigs, as indispensable large animal models in fundamental and clinical research, including the development of therapeutics for inherited and chronic disorders, and cancers.
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Papers by Karina Gutierrez