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Daniel R Arnold, Vilceu Bordignon, Réjean Lefebvre, Bruce D Murphy and Lawrence C Smith

Abnormal placental development limits success in ruminant pregnancies derived from somatic cell nuclear transfer (SCNT), due to reduction in placentome number and consequently, maternal/fetal exchange. In the primary stages of an epithelial–chorial association, the maternal/fetal interface is characterized by progressive endometrial invasion by specialized trophoblast binucleate/giant cells (TGC). We hypothesized that dysfunctional placentation in SCNT pregnancies results from aberration in expression of genes known to be necessary for trophoblast proliferation (Mash2), differentiation (Hand1), and function (IFN-τ and PAG-9). We, therefore, compared the expression of these factors in trophoblast from bovine embryos derived from artificial insemination (AI), in vitro fertilization (IVF), and SCNT prior to (day 17) and following (day 40 of gestation) implantation, as well as TGC densities and function. In preimplantation embryos, Mash2 mRNA was more abundant in SCNT embryos compared to AI, while Hand1 was highest in AI and IVF relative to SCNT embryos. IFN-τ mRNA abundance did not differ among groups. PAG-9 mRNA was undetectable in SCNT embryos, present in IVF embryos and highest in AI embryos. In postimplantation pregnancies, SCNT fetal cotyledons displayed higher Mash2 and Hand1 than AI and IVF tissues. Allelic expression of Mash2 was not different among the groups, which suggests that elevated mRNA expression was not due to altered imprinting status of Mash2. The day 40 SCNT cotyledons had the fewest number of TGC compared to IVF and AI controls. Thus, expression of genes critical to normal placental development is altered in SCNT bovine embryos, and this is expected to cause abnormal trophoblast differentiation and contribute to pregnancy loss.

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Marcelo M Seneda, Maren Godmann, Bruce D Murphy, Sarah Kimmins and Vilceu Bordignon

Follicular growth and oogenesis involve highly dynamic changes in morphogenesis, chromatin structure, and gene transcription. The tight coordination of these events leads to ovulation of a mature oocyte and formation of the luteal tissue necessary to regulate embryo implantation and development. This entire process is regulated by numerous endocrine and in situ mechanisms. The role of epigenetic mechanisms in folliculogenesis, such as the biochemical modification of the DNA packaging proteins, the histones, is not well understood. Our objective was to determine the cellular and follicular stage-specific patterns of histone H3 methylation at lysine 4 (K4) in porcine preovulatory follicles and during luteinization in pig ovaries. Ovary tissues were collected from slaughtered prepubertal and cyclic gilts at various stages of the estrous cycle, pregnancy, and from ovaries recovered from gonatropin-treated gilts at 0, 24, and 38 h post human chorionic gonadotropin (hCG) injection. Samples were fixed in 4% paraformaldehyde and processed for embedding in paraffin and sectioned using standard histological protocols. Immunofluorescent staining was performed on 3 μm thick sections. The immunostaining pattern of mono-, di-, and tri-methylated histone H3-K4 and lysine-specific demethylase 1 (LSD1, also known as KDM1 or AOF1) was assessed. Interestingly, H3-K4 mono-, di-, and tri-methylation in follicles of prepubertal gilts was specifically distributed and developmentally regulated. While granulosa cells of primary, secondary, and early antral follicles were negative for H3-K4 methylation those from large antral follicles showed a striking upregulation in the cells located in the proximity to the oocyte. Specifically, the cumulus oophorus displayed intense staining for H3-K4 methylation and signals were strongest in the granulosa cells in the inner two cell layers of the follicular wall. Although all oocytes from primary to large antral stage follicles were positive for H3-K4 mono-, di-, and tri-methylation, the patterns of distribution were altered through oocyte follicle development. H3-K4 methylation in granulosa cells was dramatically reduced as time to ovulation approached and was low to undetected at 38 h post hCG treatment. H3-K4 mono-, di-, and tri-methylation in large luteal cells increased as differentiation evolved but remained low in small luteal cells. Strikingly, LSD1 (KDM1) expression was found to be restricted to the corpus luteum. In summary, this study provides new information on histone H3-K4 methylation patterns in the oocyte and follicle during folliculogenesis, which suggests that these epigenetic markers serve an essential regulatory role during folliculogenesis.

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Rodrigo C Bohrer, Limei Che, Paulo B D Gonçalves, Raj Duggavathi and Vilceu Bordignon

Phosphorylated histone H2A.x (H2AX139ph) is a key factor for the repair of DNA double-strand breaks (DSBs) and the presence of H2AX139ph foci indicates the sites of DSBs. In this study, we characterized the presence of H2AX139ph during in vitro development of porcine embryos produced by IVF and somatic cell nuclear transfer (SCNT). Pronuclear stage embryos produced by IVF had, on average, 9.2 H2AX139ph foci per pronucleus. The number of H2AX139ph foci was higher in the 2-cell-stage embryos than in the 4-cell-stage embryos fixed at 48 h post-fertilization. The percentage of H2AX139ph-positive nuclei was higher in SCNT embryos that were activated with ionomycin (ION) alone than in those activated with ION and strontium chloride (ION+Sr2+). A negative correlation was found between the percentage of H2AX139ph-positive cells and the total number of cells per embryo in day 7 blastocysts produced by IVF or SCNT. Based on the detection of H2AX139ph foci, the findings of this study indicate that DSBs occur in a high proportion of porcine embryos produced by either IVF or SCNT; fast-cleaving embryos have fewer DSBs than slow-cleaving embryos; the oocyte activation protocol can affect DNA integrity in SCNT embryos; and better-quality blastocysts have fewer DSBs. We propose that the presence of H2AX139ph foci can be a useful marker of embryo quality.

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Lisa Dupuis, Yasmin Schuermann, Tamara Cohen, Dayananda Siddappa, Anitha Kalaiselvanraja, Melissa Pansera, Vilceu Bordignon and Raj Duggavathi

Leptin is an important hormone influencing reproductive function. However, the mechanisms underpinning the role of leptin in the regulation of reproduction remain to be completely deciphered. In this study, our objective is to understand the mechanisms regulating the expression of leptin receptor (Lepr) and its role in ovarian granulosa cells during ovulation. First, granulosa cells were collected from superovulated mice to profile mRNA expression of Lepr isoforms (LeprA and LeprB) throughout follicular development. Expression of LeprA and LeprB was dramatically induced in the granulosa cells of ovulating follicles at 4 h after human chorionic gonadotropin (hCG) treatment. Relative abundance of both mRNA and protein of CCAAT/enhancer-binding protein β (Cebpβ) increased in granulosa cells from 1 to 7 h post-hCG. Furthermore, chromatin immunoprecipitation assay confirmed the recruitment of Cebpβ to Lepr promoter. Thus, hCG-induced transcription of Lepr appears to be regulated by Cebpβ, which led us to hypothesise that Lepr may play a role during ovulation. To test this hypothesis, we used a recently developed pegylated superactive mouse leptin antagonist (PEG-SMLA) to inhibit Lepr signalling during ovulation. I.p. administration of PEG-SMLA (10 μg/g) to superovulated mice reduced ovulation rate by 65% compared with control treatment. Although the maturation stage of the ovulated oocytes remained unaltered, ovulation genes Ptgs2 and Has2 were downregulated in PEG-SMLA-treated mice compared with control mice. These results demonstrate that Lepr is dramatically induced in the granulosa cells of ovulating follicles and this induction of Lepr expression requires the transcription factor Cebpβ. Lepr plays a critical role in the process of ovulation by regulating, at least in part, the expression of the important genes involved in the preovulatory maturation of follicles.

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Gustavo Freitas Ilha, Monique T Rovani, Bernardo G Gasperin, Alfredo Quites Antoniazzi, Paulo Bayard Dias Gonçalves, Vilceu Bordignon and Raj Duggavathi

Subordinate follicles (SFs) of bovine follicular waves undergo atresia due to declining FSH concentrations; however, the signalling mechanisms have not been fully deciphered. We used an FSH-induced co-dominance model to determine the effect of FSH on signalling pathways in granulosa cells of the second-largest follicles (SF in control cows and co-dominant follicle (co-DF2) in FSH-treated cows). The SF was smaller than DF in control cows while diameters of co-DF1 and co-DF2 in FSH-treated cows were similar. The presence of cleaved CASP3 protein confirmed that granulosa cells of SFs, but not of DFs and co-DFs, were apoptotic. To determine the effect of FSH on molecular characteristics of the second-largest follicles, we generated relative variables for the second largest follicle in each cow. For this, variables of SF or co-DF2 were divided by the variables of the largest follicle DF or co-DF1 in each cow. There was higher transcript abundance of MAPK1/3 and AKT1/2/3 but lower abundance of phosphorylated MAPK3/1 in SF than co-DF2 granulosa cells. Abundance of mRNA and phosphorylated protein of STAT3 was higher in granulosa cells of control SF than FSH-treated co-DF2. SF granulosa cells had higher levels of LIFR and IL6ST transcripts, the two receptors involved in STAT3 activation. Further, lower transcript abundance of interleukin 6 receptor (IL6R), another receptor involved in STAT3 activation, indicated that STAT3 activation in SF granulosa cells could be mainly due to leukemia inhibitory factor (LIF) signalling. These results indicate that atresia due to lack of FSH is associated with activated LIF–STAT3 signalling in SF granulosa cells, as FSH treatment reversed such activation.