In vitro fertilization: productivity of donors of different bovine breeds.

The objective of the present study was to evaluate efficiency of in vitro fertilization (IVF) in Nelore, Brangus, and Girolando oocyte donors. Ovum pickup (OPU) from the donors was conducted every 15 days to assess oocyte recovery, IVF, and post-transfer pregnancy percentage. For Nelore, the mean numbers of total and viable oocytes recovered (23.5 ± 1.1 and 14.0 ± 1.0, respectively) were higher (p < 0.05) than those for Brangus (12.7 ± 1.9 and 6.6 ± 1.0, respectively) and Girolando (12.5 ± 1.4 and 6.8 ± 0.7, respectively); Brangus and Girolando did not differ from each other (p > 0.05). The percentage of blastocyst production differed (p < 0.05) between Nelore (48.4 ± 2.4%), Brangus (40.3 ± 3.6%), and Girolando (38.9 ± 2.6%), but those in Brangus and Girolando did not differ (p > 0.05). The percentage of blastocysts (transferred) that resulted in pregnancy did not differ (p > 0.05) between Nelore (45.5 ± 3.8%), Brangus (41.7 ± 4.1%), and Girolando (40.7 ± 3.7%). Of the breeds studied, Nelore donors are more efficient for IVF, but conditions of this study.

Cattle Cloning by Somatic Cell Nuclear Transfer.

Cloning by somatic cell Nuclear Transfer (SCNT) is a powerful technology capable of reprograming terminally differentiated cells to totipotency for generating whole animals or pluripotent stem cells for use in cell therapy, drug screening, and other biotechnological applications. However, the broad usage of SCNT remains limited due to its high cost and low efficiency in obtaining live and healthy offspring. In this chapter, we first briefly discuss the epigenetic constraints responsible for the low efficiency of SCNT and current attempts to overcome them. We then describe our bovine SCNT protocol for delivering live cloned calves and addressing basic questions about nuclear reprogramming. Other research groups can benefit from our basic protocol and build up on it to improve SCNT in the future. Strategies to correct or mitigate epigenetic errors (e.g., correcting imprinting loci, overexpression of demethylases, chromatin-modifying drugs) can integrate the protocol described here.

Ovarian follicular dynamics, progesterone concentrations, pregnancy rates and transcriptional patterns in Bos indicus females with a high or low antral follicle count.

We evaluated the effect of the antral follicle count (AFC) on ovarian follicular dynamics, pregnancy rates, progesterone concentrations, and transcriptional patterns of genes in Nelore cattle (Bos taurus indicus) after a timed artificial insemination (TAI) programme. Cows were separated based on the AFC, and those with a high AFC showed a larger (P < 0.0001) ovarian diameter and area than those with a very low AFC. Females with a very low AFC exhibited a larger (P < 0.01) diameter of the dominant follicle at TAI (13.6 ± 0.3 vs. 12.2 ± 0.4 mm) and a tendency (P = 0.06) to have different serum progesterone concentrations (2.9 ± 0.3 vs. 2.1 ± 0.3 ng/mL; on day 18, considering day 0 as the beginning of the synchronization protocol) than those with a high AFC. The pregnancy rate was higher (P ≤ 0.05) in animals with a very low (57.9%) and low (53.1%) AFC than in those with a high AFC (45.2%). The expression of genes related to intercellular communication, meiotic control, epigenetic modulation, cell division, follicular growth, cell maintenance, steroidogenesis and cellular stress response was assessed on day 5. In females with a low AFC, 8 and 21 genes in oocytes and cumulus cells, respectively, were upregulated (P < 0.05), while 3 and 6 genes in oocytes and cumulus cells, respectively, were downregulated. The results described here will help elucidate the differences in ovarian physiology and the reproductive success of Bos indicus females with a low or high AFC.

Catalytic inhibition of H3K9me2 writers disturbs epigenetic marks during bovine nuclear reprogramming.

Orchestrated events, including extensive changes in epigenetic marks, allow a somatic nucleus to become totipotent after transfer into an oocyte, a process termed nuclear reprogramming. Recently, several strategies have been applied in order to improve reprogramming efficiency, mainly focused on removing repressive epigenetic marks such as histone methylation from the somatic nucleus. Herein we used the specific and non-toxic chemical probe UNC0638 to inhibit the catalytic activity of the histone methyltransferases EHMT1 and EHMT2. Either the donor cell (before reconstruction) or the early embryo was exposed to the probe to assess its effect on developmental rates and epigenetic marks. First, we showed that the treatment of bovine fibroblasts with UNC0638 did mitigate the levels of H3K9me2. Moreover, H3K9me2 levels were decreased in cloned embryos regardless of treating either donor cells or early embryos with UNC0638. Additional epigenetic marks such as H3K9me3, 5mC, and 5hmC were also affected by the UNC0638 treatment. Therefore, the use of UNC0638 did diminish the levels of H3K9me2 and H3K9me3 in SCNT-derived blastocysts, but this was unable to improve their preimplantation development. These results indicate that the specific reduction of H3K9me2 by inhibiting EHMT1/2 during nuclear reprogramming impacts the levels of H3K9me3, 5mC, and 5hmC in preimplantation bovine embryos.

Sperm-borne miR-216b modulates cell proliferation during early embryo development via K-RAS.

Semen fertilizing potential is dependent upon the morphological, functional and molecular attributes of sperm. Sperm microRNAs (miRNAs) were recently shown to hold promise regarding their association with different fertility phenotypes. However, their role in fertility regulation remains to be determined. We postulated that sperm miRNAs might regulate early embryonic development. From this perspective, sperm quality and 380 sperm miRNAs were investigated in frozen-thawed semen from high (HF; 54.3 ± 1.0% pregnancy rate) and low (LF; 41.5 ± 2.3%) fertility bulls. Out of nine miRNAs that showed different levels in sperm cells, miR-216b was present at lower levels in HF sperm cells and zygotes. Among miR-216b target genes (K-RAS, BECN1 and JUN), K-RAS, related to cell proliferation, revealed a higher level in HF two-cell embryos. First cleavage rate, blastocyst cell number and division number were also higher in HF. In addition, by using a model based on polyspermy embryos, we demonstrated an increase in miR-216b levels in zygotes associated with sperm cell entry. Our results shed light on a possible mechanism of paternal contribution involving sperm-borne miR-216b that modulates levels of miR-216b in zygotes and K-RAS in two-cell embryos. This modulation might regulate early development by interfering with the first cleavage and blastocyst quality.

Metabolic gene expression and epigenetic effects of the ketone body ß-hydroxybutyrate on H3K9ac in bovine cells, oocytes and embryos.

The rapid decline in fertility that has been occurring to high-producing dairy cows in the past 50 years seems to be associated with metabolic disturbances such as ketosis, supporting the need for research to improve our understanding of the relations among the diet, metabolism and embryonic development. Recently, the ketone body ß-hydroxybutyrate (BOHB) was demonstrated to be a potent inhibitor of histone deacetylases (HDACs). Herein, we performed a series of experiments aiming to investigate the epigenetic effects of BOHB on histone acetylation in somatic cells, cumulus-oocyte complexes (COCs) and somatic cell nuclear transfer (SCNT) embryos. Treatment with BOHB does not increase histone acetylation in cells but stimulates genes associated with ketolysis and master regulators of metabolism. We further demonstrated that maturing COCs with high levels of BOHB does not affect their maturation rate or histone acetylation but increases the expression of PPARA in cumulus cells. Treatment of somatic cell nuclear transfer zygotes with BOHB causes hyperacetylation, which is maintained until the blastocyst stage, causing enhanced FOXO3A expression and blastocyst production. Our data shed light on the epigenetic mechanisms caused by BOHB in bovine cells and embryos and provide a better understanding of the connection between nutrition and reproduction.

Author Correction: Metabolic gene expression and epigenetic effects of the ketone body ß-hydroxybutyrate on H3K9ac in bovine cells, oocytes and embryos.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Development to term of cloned cattle derived from donor cells treated with valproic acid.

Cloning of mammals by somatic cell nuclear transfer (SCNT) is still plagued by low efficiency. The epigenetic modifications established during cellular differentiation are a major factor determining this low efficiency as they act as epigenetic barriers restricting reprogramming of somatic nuclei. In this regard, most factors that promote chromatin decondensation, including histone deacetylase inhibitors (HDACis), have been found to increase nuclear reprogramming efficiency, making their use common to improve SCNT rates. Herein we used valproic acid (VPA) in SCNT to test whether the treatment of nuclear donor cells with this HDACi improves pre- and post-implantation development of cloned cattle. We found that the treatment of fibroblasts with VPA increased histone acetylation without affecting DNA methylation. Moreover, the treatment with VPA resulted in increased expression of IGF2R and PPARGC1A, but not of POU5F1. However, when treated cells were used as nuclear donors no difference of histone acetylation was found after oocyte reconstruction compared to the use of untreated cells. Moreover, shortly after artificial activation the histone acetylation levels were decreased in the embryos produced with VPA-treated cells. With respect to developmental rates, the use of treated cells as donors resulted in no difference during pre- and post-implantation development. In total, five clones developed to term; three produced with untreated cells and two with VPA-treated cells. Among the calves from treated group, one stillborn calf was delivered at day 270 of gestation whereas the other one was delivered at term but died shortly after birth. Among the calves from the control group, one died seven days after birth whereas the other two are still alive and healthy. Altogether, these results show that in spite of the alterations in fibroblasts resulting from the treatment with VPA, their use as donor cells in SCNT did not improve pre- and post-implantation development of cloned cattle.

Nuclear transfer with apoptotic bovine fibroblasts: can programmed cell death be reprogrammed?

Cell death by apoptosis is considered to be irreversible. However, reports have indicated that its reversibility is possible if the cells have not yet reached the "point of no return." In order to add new information about this topic, we used cells at different moments of apoptotic process as nuclear donors in somatic cell nuclear transfer (SCNT) in order to test if programmed cell death can be reversed. Adult bovine fibroblasts were treated with 10 µM of staurosporine (STP) for 3 h and analyzed for phosphatidylserine externalization (Annexin assay) and presence of active caspase-9. Annexin-positive (Anx+) and Caspase-9-positive (Casp-9+) cells were isolated by FACS and immediately transferred into enucleated in vitro matured bovine oocytes. After STP treatment, 89.9% of cells were Anx+ (4.6% in control cells; p<0.01) and 24.9% were Casp-9+ (2.4% in control cells; p<0.01). Fusion and cleavage were not affected by the use apoptotic cells (p>0.05). Also, the use of Anx+ cells did not affect blastocyst production compared to control (26.4% vs. 22.9%, respectively; p>0.05). However, blastocyst formation was affected by the use of Casp-9+ cells (12.3%; p<0.05). These findings contribute to the idea of that apoptosis is reversible only at early stages. Additionally, we hypothesize that the "point of no return" for apoptosis may be located around activation of Caspase-9.

Treatment of nuclear-donor cells or cloned zygotes with chromatin-modifying agents increases histone acetylation but does not improve full-term development of cloned cattle.

Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatin-modifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA+TSA or HH+VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH+VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and postimplantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and postimplantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and postimplantation development of cloned cattle.