Seminal cell-free DNA as a potential marker for in vitro fertility of Nellore bulls.

PURPOSE: This study aimed to identify a marker for freezability and in vitro fertility of sperm samples before freezing. METHODS: Semen was collected from nine Nelore bulls; half of the ejaculate was used for seminal plasma cell-free DNA (cfDNA) quantification, and the other half was cryopreserved. Evaluation of sperm movement using computer-assisted semen analysis and plasma membrane integrity and stability, acrosomal integrity, apoptosis, and mitochondrial potential using flow cytometry were performed on fresh and frozen/thawed semen at 0, 3, 6, and 12 h after thawing. Frozen/thawed sperm was also used for in vitro embryo production. cfDNA was extracted from each bull, and the total DNA and number of cell-free mitochondrial DNA (cfmtDNA) copies were quantified. Semen from each animal was used for IVF, and cleavage, blastocyst formation, and cell counts were evaluated. RESULTS: Two groups were formed and compared based on the concentrations of cfDNA and cfmDNA present: low-cfDNA and high-cfDNA and low-cfmtDNA and high-cfmtDNA. Up to 12 h post-thawing, there were no differences between the groups in the majority of the sperm parameters evaluated. Cleavage, day 6 and 7 blastocyst rates, and the number of cells were higher in the high cfDNA group than in the low cfDNA group. Similar results were observed for cfmtDNA, except for the number of cells, which was similar between the groups. CONCLUSION: The concentration of cfDNA and the relative number of copies of cfmtDNA in seminal plasma cannot predict the freezability of semen but can be used to predict in vitro embryo production.

Biochemical profiling of the follicular environment to predict oocyte competence in cattle.

To identify markers of oocyte competence, we compared the biochemical characteristics of fluid and cells from follicles containing oocytes with different capacities to form an embryo. Follicles (5-6 mm) were dissected, and follicular fluid (FF), granulosa cells (GC), cumulus cells (CC) from immature and mature cumulus-oocyte-complexes (COC) were individually collected. The oocytes were matured, fertilized, and cultured individually until day 8 (D8) of development. On D8, the samples were grouped according to embryo production into those that gave rise to blastocysts (EMB) and those that did not reach the blastocyst stage (NEMB). In CCs from immature and mature COCs and GCs, expression of CASP3, SERPINE2, VCAN, LUM, FSHR, EGFR, PGR, and GHR genes was quantified. Cell-free DNA (cfDNA), progesterone, and estradiol concentrations in the FF were determined. Data were analyzed by Mann-Whitney U test (GraphPad Prism 9). GHR was highly expressed in immature CCs from the EMB group, whereas CASP3 was highly expressed in mature CCs from the NEMB group (P<0.05). During maturation, the expression of CASP3 and GHR genes increased only in the NEMB group. ART2 cfDNA was highly detected in FF of the NEMB compared to the EMB group. Progesterone concentration was similar between the groups, whereas estradiol concentration was higher (P<0.05) in the EMB than in the NEMB group. It was concluded that a higher level of GHR transcripts in immature CCs, lower CASP3 expression in CCs from matured COCs, lower levels of ART2, and higher estradiol concentrations in FF may indicate oocytes with greater potential for development.

Differentially methylated regions identified in bovine embryos are not observed in adulthood.

The establishment of epigenetic marks during the reprogramming window is susceptible to environmental influences, and stimuli during this critical stage can cause altered DNA methylation in offspring. In a previous study, we found that low levels of sulphur and cobalt (low S/Co) in the diet offered to oocyte donors altered the DNA methylome of bovine embryos. However, due to the extensive epigenetic reprogramming that occurs during embryogenesis, we hypothesized that the different methylation regions (DMRs) identified in the blastocysts may not maintain in adulthood. Here, we aimed to characterize DMRs previously identified in embryos, in the blood and sperm of adult progenies of two groups of heifers (low S/Co and control). We used six bulls and characterized the DNA methylation levels of KDM2A, KDM5A, KMT2D, and DOT1L genes. Our results showed that all DMRs analysed in both groups and tissues were hypermethylated unlike that noticed in the embryonic methylome profiles. These results suggest that embryo DMRs were reprogrammed during the final stages of de novo methylation during embryogenesis or later in development. Therefore, due to the highly dynamic epigenetic state during early embryonic development, we suggest that is essential to validate the DMRs found in embryos in adult individuals.

Differentially methylated regions identified in bovine embryos are not observed in adulthood

The establishment of epigenetic marks during the reprogramming window is susceptible to environmental influences, and stimuli during this critical stage can cause altered DNA methylation in offspring. In a previous study, we found that low levels of sulphur and cobalt (low S/Co) in the diet offered to oocyte donors altered the DNA methylome of bovine embryos. However, due to the extensive epigenetic reprogramming that occurs during embryogenesis, we hypothesized that the different methylation regions (DMRs) identified in the blastocysts may not maintain in adulthood. Here, we aimed to characterize DMRs previously identified in embryos, in the blood and sperm of adult progenies of two groups of heifers (low S/Co and control). We used six bulls and characterized the DNA methylation levels of KDM2A, KDM5A, KMT2D, and DOT1L genes. Our results showed that all DMRs analysed in both groups and tissues were hypermethylated unlike that noticed in the embryonic methylome profiles. These results suggest that embryo DMRs were reprogrammed during the final stages of de novo methylation during embryogenesis or later in development. Therefore, due to the highly dynamic epigenetic state during early embryonic development, we suggest that is essential to validate the DMRs found in embryos in adult individuals.(AU)

Using Cumulus Cell Biopsy as a Non-Invasive Tool to Access the Quality of Bovine Oocytes: How Informative Are They?

The present study aimed to determine whether cumulus cells (CC) biopsy, acquired before or after in vitro maturation (IVM), presents similar gene expression pattern and if would compromises oocyte quality. First, immature cumulus oocyte complexes (COCs) were distributed: (1) maturated in groups (control); (2) individually maturated, but not biopsied; (3) subjected to CC biopsy before maturation and individually matured; (4) individually matured and submitted to CC biopsy after maturation; (5) individually matured and CC biopsied before and after maturation. Secondly, candidate genes, described as potential markers of COCs quality, were quantified by RT-qPCR in CCs before and after IVM. After in vitro fertilization (IVF), zygotes were tracked and sorted regarding their developmental potential: fully developed to embryo, cleaved and arrested, and not-cleaved. The COC's biopsy negatively affects embryo development (p < 0.05), blastocyst cell number (p < 0.05), and apoptotic cell ratio (p < 0.05), both before and after IVM. The PTGS2, LUM, ALCAM, FSHR, PGR, SERPINE2, HAS2, and PDRX3 genes were differentially expressed (p < 0.05) on matured CCs. Only PGR gene (p = 0.04) was under-expressed on matured CCs on Not-Cleaved group. The SERPINE2 gene was overexpressed (p = 0.01) in the Cleaved group on immature CCs. In summary, none of the selected gene studies can accurately predict COC's fate after fertilization.

Dynamics of the Reproductive Changes and Acquisition of Oocyte Competence in Nelore (Bos taurus indicus) Calves during the Early and Intermediate Prepubertal Periods

The purpose of this study was to characterize the reproductive physiology, oocyte competence, and chromatin compaction in Nelore calves in the early-prepubertal period (EPP) and the intermediate-prepubertal period (IPP). Calves aged 2-5 (EPP) and 8-11 months old (IPP) were assigned to Trial 1 (morpho-physiological-endocrine evaluations, n = 8) or Trial 2 (oocyte donors, n = 8) vs. the respective control groups of cows (n = 8, each). All morphological endpoints, except the antral follicle count, increased from the EPP to the IPP. The EPP LH-FSH plasma concentrations were similar to cows, whereas LH was lower and FSH was higher in the IPP than in cows. . Cows produced more Grade I (12.9% vs. 4.1% and 1.7%) and fewer Grade III COC (30.1% vs. 44.5% and 49.0%) than the EPP and IPP calves, respectively. The IPP calves' oocyte diameter was similar to those from cows but greater than those from EPP females (124.8 ± 8.5 and 126.0 ± 7.5 µm vs. 121.3 ± 7.5 µm, respectively). The expression of the chromatin compaction-related gene HDAC3 was downregulated in calves. The proportion of the blastocyst rate to the controls was lower in EPP than in IPP calves (43.7% vs. 78.7%, respectively). Progressive oocyte competence was found during the prepubertal period, which can help to decide whether to recover oocytes from calves.

The use of insulin-transferrin-selenium (ITS), and folic acid on individual in vitro embryo culture systems in cattle.

Individual embryo culture is the only strategy that allows the tracking of embryos throughout the culture period. However, this procedure leads to lower embryo development. This study aimed to evaluate different alternatives to improve embryo development in a single in vitro production system. First, embryo production was compared between individual cultures on a 20 µL droplet and Cell-Tak® system. Then, various concentrations of folic acid were tested for use in combination with insulin-transferrin-selenium (ITS). To determine the concentration, embryos were analyzed not only by development but also by their methylation status. Finally, the supplementation of individual culture media with ITS and/or folic acid was evaluated. The results showed that embryos cultured in the Cell-Tak® system presented lower blastocyst rates than the microdroplets system. When the concentration of folic acid was tested, 20 µM and 500 µM presented a higher level of insulin-like growth factor (IGF2) DNA methylation pattern compared to control, suggesting that in vitro conditions alter DNA methylation pattern in that region and folic acid reestablishes the pattern. However, when it was used in an individual culture system, folic acid did not improve embryo development. Conversely, ITS which is composed of three important components, proved to be an alternative to individual embryo culture, improving embryo rates, showing similar rates to grouped culture embryos. Since Folic Acid change epigenetic profile, additional studies are needed to evaluate its use in IVP culture systems.

Transcriptome of D14 in vivo x in vitro bovine embryos: is there any difference?

It is well-established that in vitro culture affects quality, gene expression, and epigenetic processes in bovine embryos and that trophectoderm cells are the most susceptible to abnormalities. These changes have been reported as the main factors responsible for losses observed after transfer of in vitro-produced embryos. The present study aimed to investigate the effect of an in vitro system on bovine embryo transcriptional profiles on D14 of development. Two groups were used-one with embryos produced in vitro until D7 (day 7; VT group) and another with embryos produced in vivo by hormonal stimulation, with embryos collected on D7 (VV group). D7 embryos at similar developmental stages from both treatments were transferred to recipient uteri and recollected on D14. From D14 embryos of both treatments, trophoblast samples were removed by biopsy for sexing and transcriptome analyses. Embryos were sexed by polymerase chain reaction (PCR), and only males were used for RNA sequencing. In total, 29,005 transcripts were expressed, from which 900 were differentially expressed, but only 29 genes were significantly differentially expressed. In addition, 20 genes were found uniquely for VV and 27 for VT. These findings suggested that although the uterine environment minimized transcriptional differences, it was not able to make trophoblasts from the in vitro embryos similar to the in vivo ones. The few genes exhibiting differences are in control of important events that may be responsible for embryonic losses occurring during the first period of gestation.

Biochemical markers for pregnancy in the spent culture medium of in vitro produced bovine embryos†.

The present study aimed to identify biomarkers to assess the quality of in vitro produced (IVP) bovine embryos in the culture media. IVP embryos on Day (D) 5 of development were transferred to individual drops, where they were maintained for the last 48 h of culture. Thereafter, the medium was collected and the embryos were transferred to the recipients. After pregnancy diagnosis, the media were grouped into the pregnant and nonpregnant groups. The metabolic profiles of the media were analyzed via electrospray ionization mass spectrometry, and the concentrations of pyruvate, lactate, and glutamate were assessed using fluorimetry. The spectrometric profile revealed that the media from embryos from the pregnant group presented a higher signal intensity compared to that of the nonpregnant group; the ions 156.13 Da [M + H]+, 444.33 Da [M + H]+, and 305.97 Da [M + H]+ were identified as biomarkers. Spent culture medium from expanded blastocysts (Bx) that established pregnancy had a greater concentration of pyruvate (p = 0.0174) and lesser concentration of lactate (p = 0.042) than spent culture medium from Bx that did not establish pregnancy. Moreover, pyruvate in the culture media of Bx can predict pregnancy with 90.9% sensitivity and 75% specificity. In conclusion, we identified markers in the culture media that helped in assessing the most viable IVP embryos with a greater potential to establish pregnancy.

Electrospray mass spectrometry analysis of blastocoel fluid as a potential tool for bovine embryo selection.

PURPOSE: The aim of this study was to analyze the metabolic profiles of blastocoel fluid (BF) obtained from bovine embryos produced in vivo and in vitro. METHODS: Expanded blastocysts (20/group) that were in vitro and in vivo derived at day 7 were used. BF was collected and analyzed under direct infusion conditions using a microTOF-Q® mass spectrometer with electrospray ionization and a mass range of 50-650 m/z. RESULTS: The spectrometry showed an evident difference in the metabolic profiles of BF from in vivo and in vitro produced embryos. These differences were very consistent between the samples of each group suggesting that embryo fluids can be used to identify the origin of the embryo. Ions 453.15 m/z, 437.18 m/z, and 398.06 m/z were identified as biomarkers for the embryo's origin with 100% sensitivity and specificity. Although it was not possible to unveil the molecular identity of the differential ions, the resulting spectrometric profiles provide a phenotype capable of differentiating embryos and hence constitute a potential parameter for embryo selection. CONCLUSION: To the best of our knowledge, our results showed, for the first time, an evident difference between the spectrometric profiles of the BF from bovine embryos produced in vivo and in vitro.

Histone deacetylase inhibitor during in vitro maturation decreases developmental capacity of bovine oocytes.

This study aimed to evaluate the effect of scriptaid during pre-maturation (PIVM) and/or maturation (IVM) on developmental competence of bovine oocytes. Cumulus-oocyte complexes (COCs) were submitted to PIVM for 6 h in the presence or absence of scriptaid. COCs were distributed into five groups: T1-IVM for 22 h, T2-PIVM for 6 h and IVM for 22 h, T3-PIVM with scriptaid for 6 h and IVM for 22 h, T4-PIVM for 6 h and IVM with scriptaid for 22 h, and T5-PIVM with scriptaid for 6 h and IVM with scriptaid for 22 h. Nuclear maturation, gene expression, cumulus cells (CCs) expansion, and embryo development and quality were evaluated. At the end of maturation, all groups presented the majority of oocytes in MII (P>0.05). Only HAT1 gene was differentially expressed (P<0.01) in oocytes with different treatments. Regarding embryo development at D7, T4 (23%) and T5 (18%) had lower blastocyst rate (P<0.05) than the other treatments (T1 = 35%, T2 = 37% and T3 = 32%). No effect was observed when scriptaid in PIVM was used in less competent oocytes (P>0.05). In conclusion, presence of scriptaid in PIVM and/or IVM did not improve developmental competence or embryo quality.

DNA Methylation of the Insulin-Like Growth Factor 2-Imprinted Gene in Trophoblast Cells of Elongated Bovine Embryo: Effects of the In Vitro Culture.

DNA methylation is an essential epigenetic mark for embryo development and can be susceptible to environment factors such as in vitro conditions. The aim of this study was to verify the effect of in vitro culture until Day (D) 14 of the development on the embryo size and DNA methylation pattern of the insulin-like growth factor 2 (IGF2)-imprinted gene. To achieve this, we produced bovine embryos completely in vivo, completely in vitro, and in vitro until D7 and then in vivo up to D14. The embryos produced in in vitro were smaller than those in other two groups (p = 0.024); no differences in embryo size were observed between genders. The in vitro embryos showed a higher level of DNA methylation in the IGF2 as compared with that in the completely in vivo-produced (IVV) embryos (p = 0.009). Furthermore, totally in vitro-produced male embryos showed higher levels of DNA methylation as compared with those observed for the totally IVV male embryos (p = 0.034). No differences were observed among genders for IGF2 DNA methylation. These results showed that the window between D7 and D14 is critical for embryo development and alterations in the environmental conditions during this period can impair DNA methylation establishment of important developmental imprinted genes. This study brings unprecedented data for bovine embryos regarding the impact of the environmental conditions during the posthatching development.

The dynamics of gene expression, lipid composition and DNA methylation reprogramming are different during in vitro maturation of pig oocytes obtained from prepubertal gilts and cycling sows.

This study aimed to characterize the gene expression, lipid composition and DNA methylation reprogramming during in vitro maturation (IVM) of pig oocytes with different developmental competencies. We used prepubertal gilts and cycling sows as a model to obtain oocytes with different levels of competency. We found that genes involved in lipid metabolism, SLC27A4, CPT2 and PLIN2, and DNA methylation, DNMT3A, TET1 and TET3, possessed altered transcript expression levels during IVM. Specifically, SLC27A4 mRNA (p = 0.05) increased in oocytes from cycling females, whereas CPT2 (p = 0.05), PLIN2 (p = 0.02) and DNMT3A (p = 0.02) increased in oocytes from prepubertal females during IVM. Additionally, TET3 mRNA increased during IVM in oocytes from prepubertal (p = 0.0005) and cycling females (p = 0.02). The TET1 transcript decreased (p = 0.05) during IVM in oocytes from cycling sows. Regarding lipid composition, mass spectrometry revealed a cluster of ions, with molecular masses higher than m/z 700, which comprises a group of complex phospholipids, was identified in all groups of oocytes, except in those from prepubertal gilts. With respect to DNA methylation reprogramming, it was noted that the less competent oocytes were not able to reprogramme the XIST gene during IVM. We conclude that the maternal mRNA store, lipid composition and epigenetic reprogramming are still being established during maturation and are related to oocyte competence. In addition, we propose that the methylation pattern of the XIST may be used as molecular marker for oocyte competence in pigs.

DNA methylation and functional characterization of the XIST gene during in vitro early embryo development in cattle.

XIST, in association with the shorter ncRNA RepA, are essential for the initiation of X chromosome inactivation (XCI) in mice. The molecular mechanisms controlling XIST and RepA expression are well characterized in that specie. However, little is known in livestock. We aimed to characterize the DNA methylation status along the 5' portion of XIST and to characterize its transcriptional profile during early development in cattle. Three genomic regions of XIST named here as promoter, RepA and DMR1 had their DNA methylation status characterized in gametes and embryos. Expression profile of XIST was evaluated, including sense and antisense transcription. Oocytes showed higher levels of methylation than spermatozoa that was demethylated. DMR1 was hypermethylated throughout oogenesis. At the 8-16-cell embryo stage DMR1 was completed demethylated. Interestingly, RepA gain methylation during oocyte maturation and was demethylated at the blastocyst stage, later than DMR1. These results suggest that DMR1 and RepA are transient differentially methylated regions in cattle. XIST RNA was detected in matured oocytes and in single cells from the 2-cell to the morula stage, confirming the presence of maternal and embryonic transcripts. Sense and antisense transcripts were detected along the XIST in blastocyst. In silico analysis identified 63 novel transcript candidates at bovine XIST locus from both the plus and minus strands. Taking together these results improve our understanding of the molecular mechanisms involved in XCI initiation in cattle. This information may be useful for the improvement of assisted reproductive technologies in livestock considering that in vitro conditions may impair epigenetic reprogramming.

Use of trichostatin A alters the expression of HDAC3 and KAT2 and improves in vitro development of bovine embryos cloned using less methylated mesenchymal stem cells.

The aim of this work was to investigate the methylation and hydroxymethylation status of mesenchymal stem cells (MSC) from amniotic fluid (MSC-AF), adipose tissue (MSC-AT) and fibroblasts (FIB-control) and to verify the effect of trichostatin A (TSA) on gene expression and development of cloned bovine embryos produced using these cells. Characterization of MSC from two animals (BOV1 and BOV2) was performed by flow cytometry, immunophenotyping and analysis of cellular differentiation genes expression. The cells were used in the nuclear transfer in the absence or presence of 50 nM TSA for 20 hr in embryo culture. Expression of HDAC1, HDAC3 and KAT2A genes was measured in embryos by qRT-PCR. Methylation results showed difference between animals, with MSC from BOV2 demonstrating lower methylation rate than BOV1. Meanwhile, MSC-AF were less hydroxymethylated for both animals. MSC-AF from BOV2 produced 44.92 ± 8.88% of blastocysts when embryos were exposed to TSA and similar to embryo rate of MSC-AT also treated with TSA (37.96 ± 15.80%). However, when methylation was lower in FIB compared to MSC, as found in BOV1, the use of TSA was not sufficient to increase embryo production. MSC-AF embryos expressed less HDAC3 when treated with TSA, and expression of KAT2A was higher in embryos produced with all MSC and treated with TSA than embryos produced with FIB. The use of MSC less methylated and more hydroxymethylated in combination with embryo incubation with TSA can induce lower expression of HDAC3 and higher expression of KAT2A in the embryos and consequently improve bovine embryo production.

Biopsy of bovine embryos produced in vivo and in vitro does not affect pregnancy rates.

Assisted reproductive techniques have significantly contributed to animal breeding programs. Similarly, genomics has provided important information and tools to improve the accuracy of selection. However, the greatest benefits of those tools can only be expected when they are combined, allowing animals to be selected accurately early in life. Therefore, obtaining DNA samples from embryos without compromising their viability is essential for the consolidation of preimplantation genomic selection. We aimed to evaluate the effect on the gestation rate of conducting a biopsy of in vivo (VV) and in vitro-produced (IVP) bovine embryos. The VV and IVP embryos were distributed into two groups: VV-B (biopsied embryos; n = 380) and VV-C (intact embryos-controls; n = 229) and IVP-B (biopsied embryos; n = 91) and IVP-C (intact embryos-controls; n = 227), respectively. After biopsy, embryos from both groups VV-B and IVP-B were cultured for an additional 3 hours before being transferred to synchronized recipients. To evaluate the quality of the DNA obtained in the biopsies, this was used to determine the sex of embryos by polymerase chain reaction. No effect (P > 0.05) of the biopsy was observed for any of the treatments, the pregnancy rate at D 60 post-transfer being similar for VV-B: 206/380 (54.21%) and VV-C: 128/229 (55.89%) and for IVP-B: 24/91 (26.37%) and IVP-C: 45/227 (19.82%). Also, no effect (P > 0.05) of the embryo's stage of development was detected on percentage of pregnant recipients when in vitro embryos were transferred. From the biopsies analyzed, about 90% had the sex determined, confirming that DNA was there and it was efficiently amplified. The results indicated that biopsy does not affect the viability of IVV and IVP bovine embryos and can be used in commercial programs to associate assisted reproductive technologies with genomic selection.

Effect of vitrification using the Cryotop method on the gene expression profile of in vitro-produced bovine embryos.

The present study analyzed the changes in gene expression induced by the Cryotop vitrification technique in bovine blastocyst-stage embryos, using Agilent EmbryoGENE microarray slides. Bovine in vitro-produced embryos were vitrified and compared with nonvitrified (control) embryos. After vitrification, embryos were warmed and cultured for an additional 4 hours. Survived embryos were used for microarray analysis and quantitative polymerase chain reaction (qPCR) quantification. Survival rates were higher (P < 0.05) in the control embryos (100%) than in the vitrified embryos (87%). Global gene expression analysis revealed that only 43 out of 21,139 genes exhibited significantly altered expression in the vitrified embryos compared to the control embryos, with a very limited fold change (P < 0.05). A total of 10 genes were assessed by qPCR. Only the FOS-like antigen 1 (FOSL1) gene presented differential expression (P < 0.05) according to both the array and qPCR methods, and it was overexpressed in vitrified embryos. Although, the major consequence of vitrification seems to be the activation of the apoptosis pathway in some cells. Indeed, FOSL1 is part of the activating protein 1 transcription factor complex and is implicated in a variety of cellular processes, including proliferation, differentiation, and apoptosis. Therefore, our results suggest that a limited increase in the rate of apoptosis was the only detectable response of the embryos to vitrification stress.

Characterization of the IGF2 Imprinted Gene Methylation Status in Bovine Oocytes during Folliculogenesis.

DNA methylation reprogramming occurs during mammalian gametogenesis and embryogenesis. Sex-specific DNA methylation patterns at specific CpG islands controlling imprinted genes are acquired during this window of development. Characterization of the DNA methylation dynamics of imprinted genes acquired by oocytes during folliculogenesis is essential for understanding the physiological and genetic aspects of female gametogenesis and to determine the parameters for oocyte competence. This knowledge can be used to improve in vitro embryo production (IVP), specifically because oocyte competence is one of the most important aspects determining the success of IVP. Imprinted genes, such as IGF2, play important roles in embryo development, placentation and fetal growth. The aim of this study was to characterize the DNA methylation profile of the CpG island located in IGF2 exon 10 in oocytes during bovine folliculogenesis. The methylation percentages in oocytes from primordial follicles, final secondary follicles, small antral follicles, large antral follicles, MII oocytes and spermatozoa were 73.74 ± 2.88%, 58.70 ± 7.46%, 56.00 ± 5.58%, 65.77 ± 5.10%, 56.35 ± 7.45% and 96.04 ± 0.78%, respectively. Oocytes from primordial follicles showed fewer hypomethylated alleles (15.5%) than MII oocytes (34.6%) (p = 0.039); spermatozoa showed only hypermethylated alleles. Moreover, MII oocytes were less methylated than spermatozoa (p<0.001). Our results showed that the methylation pattern of this region behaves differently between mature oocytes and spermatozoa. However, while this region has a classical imprinted pattern in spermatozoa that is fully methylated, it was variable in mature oocytes, showing hypermethylated and hypomethylated alleles. Furthermore, our results suggest that this CpG island may have received precocious reprogramming, considering that the hypermethylated pattern was already found in growing oocytes from primordial follicles. These results may contribute to our understanding of the reprogramming of imprinted genes during bovine oogenesis.

Padrão de metilação dos genes xist e igf2 em ovocitos de folículos antrais de vacas nelore

Modificações epigenéticas são eventos que controlam a expressão diferencial de genes, sendo a metilação do DNA um dos mais conhecidos, importante para a reprogramação epigenética durante a gametogênese. Entender como isso ocorre na ovogênese é importante para criar parâmetros para a competência ovocitária com o intuito de melhorar a produção in vitro de embriões. Nesse trabalho objetivou-se avaliar o padrão de metilação em ovócitos de duas categorias de folículos antrais: entre 1 e 3 mm e maiores que 6 mm. Para isso foram escolhidas duas ilhas CpGs, uma no éxon 1 do gene XIST que está relacionado com a inativação do cromossomo X em fêmeas, e outra no último éxon do gene IGF2 envolvido no desenvolvimento embrionário e placentação. Os ovócitos foram obtidos a partir de ovários de vacas Nelore provenientes de abatedouros usando o tissue chopper e sucessivas pipetagens para o isolamento dos ovócitos. A classificação dos ovócitos foi realizada de acordo com o seu diâmetro. O DNA extraído dos ovócitos foi tratado com bissulfito de sódio e os genes de interesse foram amplificados através de PCR nested. Os produtos purificados de gel de agarose 2% foram utilizados clonagem em células DH5α. Os clones foram sequenciados usando a metodologia dideoxi e analisados no programa BiQ Analyzer utilizando sequências controle do “GenBank”. Apenas sequências com mais de 90% de identidade e de conversão pelo bissulfito de sódio foram consideradas para análises. Os resultados mostraram que os ovócitos de folículos entre 1 e 3 mm e maiores que 6 mm possuem padrão de metilação iguais, tanto para o gene XIST (89,5% e 92,7%, respectivamente) quanto para o gene IGF2 (73,4% e 61,5%, respectivamente). Esses dados sugerem que, pelo menos para as regiões estudadas, o padrão de metilação já está estabelecido a partir da fase antral do folículo ovariano em ovócitos imaturos.(AU)

Padrão de metilação dos genes xist e igf2 em ovocitos de folículos antrais de vacas nelore

Modificações epigenéticas são eventos que controlam a expressão diferencial de genes, sendo a metilação do DNA um dos mais conhecidos, importante para a reprogramação epigenética durante a gametogênese. Entender como isso ocorre na ovogênese é importante para criar parâmetros para a competência ovocitária com o intuito de melhorar a produção in vitro de embriões. Nesse trabalho objetivou-se avaliar o padrão de metilação em ovócitos de duas categorias de folículos antrais: entre 1 e 3 mm e maiores que 6 mm. Para isso foram escolhidas duas ilhas CpGs, uma no éxon 1 do gene XIST – que está relacionado com a inativação do cromossomo X em fêmeas, e outra no último éxon do gene IGF2 – envolvido no desenvolvimento embrionário e placentação. Os ovócitos foram obtidos a partir de ovários de vacas Nelore provenientes de abatedouros usando o tissue chopper e sucessivas pipetagens para o isolamento dos ovócitos. A classificação dos ovócitos foi realizada de acordo com o seu diâmetro. O DNA extraído dos ovócitos foi tratado com bissulfito de sódio e os genes de interesse foram amplificados através de PCR nested. Os produtos purificados de gel de agarose 2% foram utilizados clonagem em células DH5α. Os clones foram sequenciados usando a metodologia dideoxi e analisados no programa BiQ Analyzer utilizando sequências controle do “GenBank”. Apenas sequências com mais de 90% de identidade e de conversão pelo bissulfito de sódio foram consideradas para análises. Os resultados mostraram que os ovócitos de folículos entre 1 e 3 mm e maiores que 6 mm possuem padrão de metilação iguais, tanto para o gene XIST (89,5% e 92,7%, respectivamente) quanto para o gene IGF2 (73,4% e 61,5%, respectivamente). Esses dados sugerem que, pelo menos para as regiões estudadas, o padrão de metilação já está estabelecido a partir da fase antral do folículo ovariano em ovócitos imaturos.