Rodrigo C. Bohrer

Inhibition of histone deacetylases enhances DNA damage repair in SCNT embryos

Recent studies have shown that DNA damage affects embryo development and also somatic cell reprogramming into induced pluripotent stem (iPS) cells. It has been also shown that treatment with histone deacetylase inhibitors (HDACi) improves development of embryos produced by somatic cell nuclear transfer (SCNT) and enhances somatic cell reprogramming. There is evidence that increasing histone acetylation at the sites of DNA double-strand breaks (DSBs) is critical for DNA damage repair. Therefore, we hypothesized that HDACi treatment enhances cell programming and embryo development by facilitating DNA damage repair. To test this hypothesis, we first established a DNA damage model wherein exposure of nuclear donor cells to ultraviolet (UV) light prior to nuclear transfer reduced the development of SCNT embryos proportional to the length of UV exposure. Detection of phosphorylated histone H2A.x (H2AX139ph) foci confirmed that exposure of nuclear donor cells to UV light for 10 s was sufficient to increase DSBs in SCNT embryos. Treatment with HDACi during embryo culture increased development and reduced DSBs in SCNT embryos produced from UV-treated cells. Transcript abundance of genes involved in either the homologous recombination (HR) or nonhomologous end-joining (NHEJ) pathways for DSBs repair was reduced by HDACi treatment in developing embryos at day 5 after SCNT. Interestingly, expression of HR and NHEJ genes was similar between HDACi-treated and control SCNT embryos that developed to the blastocyst stage. This suggested that the increased number of embryos that could achieve the blastocyst stage in response to HDACi treatment have repaired DNA damage. These results demonstrate that DNA damage in nuclear donor cells is an important component affecting development of SCNT embryos, and that HDACi treatment after nuclear transfer enhances DSBs repair and development of SCNT embryos.

Expression and molecular consequences of inhibition of estrogen receptors in granulosa cells of bovine follicles

BackgroundEstradiol (E2) receptors mediate E2 effects on cell proliferation and apoptosis under normal and pathological conditions. However, the mechanisms involved in E2 signaling are not completely understood. The objectives in this study were to evaluate the expression of estrogen receptors (ESRs) during follicular selection in cattle, and the effect of intrafollicular injection of fulvestrant (an antagonist of ESRs) on follicular development and transcript abundance in granulosa cells.MethodsGranulosa cells were obtained from the two largest follicles around follicular deviation, after FSH treatment and after intrafollicular injection of fulvestrant. Ovarian follicular dynamics monitored by ultrasonography and quantitative real time PCR were used to validate the in vivo model and investigate the effects of FSH supplementation or ESR blockade on mRNA expression of estradiol-related genes.ResultsESR1 and ESR2 were expressed in granulosa cells of both dominant (F1) and subordinate (F2) follicles, but their transcripts levels were higher in F1 than F2 after follicular deviation. FSH treatment maintained mRNA levels of both ESR1 and ESR2 in F2 follicles at similar levels observed in F1 follicles. Intrafollicular injection of 100 μM fulvestrant inhibited follicular growth and decreased CYP19A1 mRNA levels. Transcript levels for both ESR1 and ESR2 were not affected by fulvestrant injection. Analyses of FSH-regulated genes revealed that ESRs inhibition in the dominant follicle decreased the transcript levels of the GJA1 but not those of PRKAR2B, MRO or LRP11 genes.ConclusionsOur findings indicate that: both ESR1 and ESR2 are regulated during follicular deviation and dominance in cattle and in response to FSH treatment, and ESRs are required for normal gene expression and development of the dominant follicle. Furthermore, we have validated an in vivo model to study estrogen signaling during follicular development that allows paracrine signaling between different follicular cells in a physiological endocrine environment.

The Incidence of DNA Double-Strand Breaks Is Higher in Late-Cleaving and Less Developmentally Competent Porcine Embryos

ABSTRACT Studies in different species, including human, mice, bovine, and swine, demonstrated that early-cleaving embryos have higher capacity to develop to the blastocyst stage and produce better quality embryos with superior capacity to establish pregnancy than late-cleaving embryos. It has also been shown that experimentally induced DNA damage delays embryo cleavage kinetics and reduces blastocyst formation. To gain additional insights into the effects of genome damage on embryo cleavage kinetics and development, the present study compared the occurrence of DNA double-strand breaks (DSBs) with the expression profile of genes involved in DNA repair and cell cycle control between early- and late-cleaving embryos. Porcine oocytes matured in vitro were activated, and then early-cleaving (before 24 h) and late-cleaving (between 24 and 48 h) embryos were identified and cultured separately. Developing embryos, on Days 3, 5, and 7, were used to evaluate the total cell number and presence of DSBs (by counting the number of immunofluorescent foci for phosphorylated histone H2A.x [H2AX139ph] and RAD51 proteins) and to quantify transcripts of genes involved in DNA repair and cell cycle control by quantitative RT-PCR. Early-cleaving embryos had fewer DSBs, lower transcript levels for genes encoding DNA repair and cell cycle checkpoint proteins, and more cells than late-cleaving embryos. Interestingly, at the blastocyst stage, embryos that developed from early- and late-cleaving groups had similar number of DSBs as well as transcript levels of genes induced by DNA damage. This indicates that only embryos with less DNA damage and/or superior capacity for DNA repair are able to progress to the blastocyst stage. Collectively, findings in this study revealed a negative correlation between the occurrence of DSBs and embryo cleavage kinetics and embryo developmental capacity to the blastocyst stage.

Relief of endoplasmic reticulum stress enhances DNA damage repair and improves development of pre-implantation embryos

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 the importance of stress coping responses in early developing embryos, and that reduction of ER stress is a potential means to improve embryo quality and developmental competence.

Natriuretic peptide system regulation in granulosa cells during follicle deviation and ovulation in cattle

Natriuretic peptides (NPs) are known to regulate reproductive events in polyovulatory species, but their function and regulation in monovulatory species remain to be fully characterized. Using a well-established in vivo model, we found that bovine granulosa cells from follicles near the deviation stage express mRNA for the three NP receptors (NPR1, NPR2 and NPR3), but not for NP precursors (NPPA, NPPB and NPPC). The abundance of NPR3 mRNA was higher in dominant compared to subordinate follicles at the expected time of follicular deviation. After deviation, mRNA for all NP receptors was significantly more abundant in the dominant follicle. Intrafollicular inhibition of oestrogen receptors downregulated NPR1 mRNA in dominant follicles. In granulosa cells from preovulatory follicles, NPPC mRNA increased at 3 and 6xa0h after systemic GnRH treatment, but decreased at 12 and 24xa0h to similar levels observed in samples collected at 0xa0h. After GnRH treatment, NPR1 mRNA was upregulated at 24xa0h, NPR3 mRNA gradually decreased after 3xa0h, while NPR2 mRNA was not regulated. The mRNA expression of the enzyme FURIN increased at 24xa0h after GnRH treatment. These findings revealed that the expression of mRNA encoding important components of the NP system is regulated in bovine granulosa cells during follicular deviation and in response to GnRH treatment, which suggests a role of NP system in the modulation of these processes in monovulatory species.

The effect of age and length of gonadotropin stimulation on the in vitro embryo development of Holstein calf oocytes

The use of oocytes recovered from prepubertal donors for inxa0vitro 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; 36-42xa0h), Long (LT; ≥72xa0h) and No Treatment (NT) prior to laparoscopic ovum pick-up (LOPU). Our results show that the LT significantly improved the proportion of large follicles (>5xa0mm diameter) present in the ovary (LT 34.0% vs. ST 11.2% vs. NT 2.4%, Pxa0<xa00.05), as well as the percentage of good-quality cumulus oocyte complexes (COCs) recovered (LT 95.3xa0±xa018% vs. ST 85.4xa0±xa022% vs. NT 82.2xa0±xa014%, Pxa0<xa00.05) and blastocyst rate (LT 36.7xa0±xa026% vs. ST 18.3xa0±xa015% vs. NT 16.7xa0±xa09%, Pxa0<xa00.05). Recovery rate was affected by treatment (LT 70.4xa0±xa025 vs. ST 85.4xa0±xa029 vs. NT 72.7xa0±xa023, Pxa0<xa00.05). To assess the impact of age, data was grouped into <100 days (A), 100-130 days (B) and >130 days (C) of age at LOPU. We found that as animals got older, although the average number of COCs per donor per LOPU declined (A: 17.5xa0±xa011 vs. B: 14.7xa0±xa07 vs. C: 11.9xa0±xa08), the blastocyst rate increased (A: 12.8xa0±xa020% vs. B: 17.1xa0±xa021% vs. C: 21.8xa0±xa025%, Pxa0<xa00.05). We also evaluated the incidence of polyspermy and confirmed it is a critical limitation for IVF in calf oocytes. The incidence of polyspermy was unaffected by gonadotropin treatment, but significantly decreased with age. The capacity for full development to term of inxa0vitro produced embryos from calf oocytes was tested by embryo transfer into 21 synchronized adult recipients, which resulted in 13 pregnancies (62%), full development to term and healthy calves born. Finally, the study allowed evaluating the safety of the procedure since, on average, each animal was subjected to 8 LOPU procedures over a period of 4 months. Our results showed that the procedure is safe (no incidents during laparoscopy), and was not harmful for the reproductive future of the animals, as those that were bred became pregnant after reaching sexual maturity.

The components of the angiotensin-(1-7) system are differentially expressed during follicular wave in cattle:

Introduction: This study was based on the hypothesis that some components of the angiotensin-(1-7) (Ang-(1-7)) system are differentially expressed during follicular development and can be involved in the follicular health/atresia transition in bovine. Material and methods: The largest (F1) and second largest follicles (F2) were collected from cows before (Day 2), during (Day 3), or after (Day 4) the expected moment of follicular deviation. In the second experiment, F1 was induced to atresia through intrafollicular injection of fulvestrant (estrogen receptor-antagonist) and, in both experiments, mRNA expression of the Mas receptor, ACE2, NEP, and PEP was evaluated in the granulosa and theca cells. Results: The mRNA expression of Mas receptor was upregulated in the granulosa cells of F2 after the establishment of follicular deviation, while PEP mRNA increased during and after the deviation process. The mRNA expression of ACE2 was upregulated in the granulosa cells of F1 during and after the follicular deviation. The mRNA expression of NEP was not regulated in F1 and F2. Mas receptor expression increased in the F1 induced to atresia. Conclusions: mRNA for Mas receptor, ACE2, and PEP are differentially expressed in granulosa cells throughout follicular development and the Mas receptor can be involved with the establishment of follicular dominance.

Interval of gonadotropin administration for in vitro embryo production from oocytes collected from Holstein calves between 2 and 6 months of age by repeated laparoscopy

Laparoscopic Ovum Pick-Up (LOPU) in calves followed by inxa0vitro 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 2-6u202fmonthsu202fat 2-3 weeks interval. In all cases, the animals received a CIDR 5 days prior to LOPU and were gonadotropin-stimulated starting at 72u202fh before LOPU using one of three protocols that were rotated twice among the animals during the study. Calves were injected with FSH every 12u202fh (FSH12h), or every 8u202fh (FSH8h) or every 8u202fh untilxa0-36u202fh 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 (35.7u202f±u202f16 vs. 38.5u202f±u202f25 vs. 31.1u202f±u202f22), mean oocytes recovered (26.5u202f±u202f14 vs. 21.6u202f±u202f10 vs. 19.4u202f±u202f14) and cleavage rate (66.0u202f±u202f14 vs. 61.1u202f±u202f11 vs. 72.2u202f±u202f8), for FSH12h, FSH8h and FSH8h-eCG, respectively. However, FSH8h-eCG resulted in a significantly higher rate of transferable embryos (17.5u202f±u202f8%) compared with FSH12h (8.9u202f±u202f5%, Pu202f<u202f0.05). Oocytes from follicles of ≥5u202fmm in diameter yielded a higher rate (Pu202f<u202f0.05) of development to the blastocyst stage (13.8%) than those collected from <5u202fmm follicles (6.8%). Animal age, by comparing animals at <100, 101 to 130 andu202f>u202f130 days of age, did not affect the mean number of follicles (34.2u202f±u202f15 vs. 39.3u202f±u202f26 vs. 31.6u202f±u202f25), the mean number of oocytes recovered (21.2u202f±u202f10 vs. 24.5u202f±u202f15 vs. 22.6u202f±u202f17), and the cleavage rate (68.6u202f±u202f11 vs. 61.7u202f±u202f12 vs. 70.7u202f±u202f10%), respectively. However, animals in the older age range had significantly higher development to the blastocyst stage (19.9u202f±u202f6 vs. 9.5u202f±u202f8%, Pu202f<u202f0.01) and better embryo quality, as evidenced by higher average cell numbers (119.1u202f±u202f47 vs. 91.5u202f±u202f25, Pu202f<u202f0.05) compared with those in the lower age. Finally, we tested the benefits of relieving endoplasmic reticulum stress by supplementing the culture medium with 50u202fμM tauroursodeoxycholic acid (TUDCA) and found a numerically higher rate of development to the blastocyst stage (21.1u202f±u202f8 vs. 18.6u202f±u202f4%), but not statistically different, compared with control culture. Overall, our findings indicate that a significant number of transferable embryos (range 10-30) can be produced from Holstein calves before they reach 6 months of age.

Granulosa cells of prepubertal cattle respond to gonadotropin signaling and upregulate genes that promote follicular growth and prevent cell apoptosis: MICHALOVIC et al.

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), and preventing cell apoptosis ( XIAP), and by decreasing mRNA levels of genes promoting progesterone production ( StAR and HSD3B1). This study also revealed that the relative mRNA abundance of FOXO1 in GCs is associated with oocyte competence to support embryo development to the blastocyst stage in prepubertal Holstein heifers.

Double-strand DNA breaks are mainly repaired by the homologous recombination pathway in early developing swine embryos

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 ATM alone resulted in a higher incidence of DSBs, lower development and embryo quality, and increased mRNA abundance of genes that are involved in either repair pathway. These findings indicate that HR is the main pathway responsible for the promotion of DSB repair in early developing embryos, and that ATM seems to be more important than ATR in the regulationofthe HR pathwayinmammalianembryos.— Bohrer, R. C., Dicks, N., Gutierrez, K., Duggavathi, R., Bordignon, V. Double‐strand DNA breaks are mainly repaired by the homologous recombination pathway in early developing swine embryos. FASEB J. 32, 1818–1829 (2018). www.fasebj.org