S. Ledda

The subcortical maternal complex: multiple functions for one biological structure?

The subcortical maternal complex (SCMC) is a multiprotein complex uniquely expressed in mammalian oocytes and early embryos, essential for zygote progression beyond the first embryonic cell divisions. Similiar to other factors encoded by maternal effect genes, the physiological role of SCMC remains unclear, although recent evidence has provided important molecular insights into different possible functions. Its potential involvement in human fertility is attracting increasing attention; however, the complete story is far from being told. The present mini review provides an overview of recent findings related to the SCMC and discusses its potential physiological role/s with the aim of inspiring new directions for future research.

In vitro Developmental Competence of Adult Sheep Oocytes Treated with Roscovitine

The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 μm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos.

Vasoactive intestinal peptide influences hatching of ovine blastocysts

Expanded blastocysts collected from superovulated Sarda ewes were divided at random into four groups for culture in a simple medium that does not support blastocyst hatching (CZB) or a complex medium that is permissive to hatching (TCM 199), with or without vasoactive intestinal peptide (VIP), a known embryo mitogenic peptide. Plasminogen activator (PA) secretion after 24 h of culture, and the number of cells, diameter of blastocysts and hatching rate after 48 h of culture were compared. The results showed an increase in hatching rate (78.6 v. 6.7%; P<0.01), diameter and number of cells (220.89 v. 210.44 microm, P<0.01 and 246 v. 232, P<0.01 respectively) and caseinolytic areas (1.33 v. 0.92 cm, P<0.01) of blastocysts cultured in TCM 199 compared with those cultured in CZB. Supplementation of the culture media with VIP increased these parameters in CZB (P<0.01) and partially in TCM 199. In particular, cell number, diameter and PA activity were significantly higher (P<0.01) after culture with VIP in both media. Immunoneutralization of exogenous VIP in culture with anti-VIP antibody caused a decrease in the hatching rate (P<0.01) of embryos cultured in medium with VIP, similar to the rate in unsupplemented CZB (P<0.01). These results suggest a receptor-mediated response. In immunohistochemical studies, VIP was shown to bind receptors in hatched blastocysts demonstrating the VIP-receptor interaction, and VIP receptors of approximately 150 kDa were revealed by electrophoretic studies. In conclusion, ovine preimplantation embryos exhibit VIP receptors, providing a basis for a receptor-mediated influence of VIP in the hatching of ovine blastocysts.

196 USE OF A MICRO-BIOREACTOR TO PROMOTE 3-DIMENSIONAL CELL REARRANGEMENT AND INDUCE, MAINTAIN, AND STABILIZE HIGH PLASTICITY IN EPIGENETICALLY ERASED FIBROBLASTS

Development and cell differentiation are driven by complex epigenetic mechanisms that regulate chromatin structure and specific gene transcription programs. We recently demonstrated that it is possible to modify the epigenetic signature of terminally differentiated cells, switching their phenotype into one of higher plasticity, through the use of molecules that remove epigenetic marks from DNA and histones (Pennarossa et al. 2013 Proc. Natl. Acad. Sci. 110, 8948-8953; Brevini et al. 2014 Stem Cell Rev. 10, 633-642). Here we drive mammalian fibroblasts into a high plasticity state using the epigenetic eraser, 5-aza-cytidine (5-aza-CR), and investigate whether the simultaneous use of a micro-bioreactor culture system is able to promote three-dimensional (3D) cell rearrangement, boost the induction of high plasticity, and stably maintain it. To this purpose, fibroblasts were either plated on plastic dishes (Group A) or encapsulated in a liquid marble micro-bioreactor (polytetrafluoroethylene powder; Sigma 430935, St. Louis, MO; Group B). Both groups were erased with 5-aza-CR and cultured in embryonic stem cell medium for 28 days. Morphological analysis was carried out for the entire length of the experiment. The OCT4, NANOG, and REX1 expression levels were assessed by real-time PCR at different time points. Exposure to 5-aza-CR induced a dramatic change in morphology in Group A fibroblasts. Cells became rounded, with larger and granulated nuclei and retained a monolayer distribution for the entire length of the experiment. The same changes in cell and nuclear morphology were observed also in cells encapsulated in liquid marble (Group B). In addition, these cells formed 3D spherical structures that were stably maintained until Day 28. These morphological rearrangements were accompanied by the active expression of the pluripotency markers, OCT4, NANOG, and REX1, in both groups. However, while Group A cells progressively down-regulated their expression by Day 6, Group B cells steadily transcribed these genes until Day 28, when cultures were arrested. Altogether, the data confirm that epigenetic erasing induces a high plasticity state in terminally differentiated fibroblasts with the expression of pluripotency related genes. Striking morphological changes accompanied the removal of epigenetic marks. These were influenced by the use of an adequate 3D in vitro culture system, with the induction of distinctive cell rearrangements and the formation of spherical structures that boosted and maintained cell plasticity. These results suggest a correlation between the mechanotransduction pathways induced by the micro-bioreactor culture system and the epigenetic regulation of cell phenotype.

258 EXPRESSION PATTERN OF THE MATERNAL FACTOR ZYGOTE ARREST 1 (ZAR1) GENE IN OVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

Zygote arrest 1 (ZAR1) is an ovary-specific maternal factor that plays essential roles during the oocyte-to-embryo transition. It is evolutionary conserved in vertebrates, and the protein is characterized by the presence of a homeobox zing finger domain, suggesting its role in transcription regulation. Discovered in mice (Wu et al. 2003 Nat. Genet. 33, 187-191), its expression pattern has been analyzed in oocytes and pre-implantation embryos in mice, cattle, and pig (Pennetier et al. 2004 Biol. Reprod. 71, 1359-1366; Brevini et al. 2004 Mol. Reprod. Dev. 69, 375-380; Reprod. Biol. Endocrinol. 21, 4-12). Because no information on Zar1 ortholog in the ovine species is available, the aim of this study was to assess its presence and to analyze its expression pattern in oocytes and pre-implantation embryos. Reverse transcription-polymerase chain reaction (RT-PCR) was performed in germinal vesicle (GV) and IVM metaphase II (MII) oocytes, and, following IVFC, in 2- (2C), 4- (4C), 8- (8C), and 12-16- (12C-16C) cell embryos, morulae, and blastocysts. Primers were designed on the basis of bovine and swine conserved sequences and contained an intron-spanning region. The PCR product was sequenced and the alignment, performed with BLAST (www.ncbi.nlm.nih.gov/BLAST/), confirmed its homology with the Zar1 orthologous genes present in public databases, sharing 98%, 92%, and 89% nucleotide identity with the bovine, porcine, and human sequences, respectively. Real-time PCR revealed that Zar1 is present at its highest level in the GV oocyte, has a major decrease at the MII stage (around 10-fold), remains constant in the embryo first cleavage steps (2C-4C), drops again at the 8C stage (around 30-fold), and disappears in morulae and blastocysts. Our data demonstrate that a Zar1 ortholog is present in the ovine species. Results are also in accordance with the expression patterns characterized in other mammalian species and suggest that Zar1 transcript in the ovine oocyte may be necessary for normal female reproduction in the transition from oocyte to embryonic life.

321 Oocyte diameter influences the meiotic resumption and progression induced by okadaic acid in dog.

The acquisition of meiotic competence, in the bitch as in many other mammalian species, is related to the oocyte diameter. This study was designed to determine the effect of okadaic acid (OA), a potent inhibitor of seronine/threonine 1 and 2A phosphatases, on meiotic resumption and progression in canine oocytes with different diameters. In two experiments, healthy cumulus-oocytes complexes were collected from ovaries of bitches at various stages of the estrous cycle and divided, by diameters, into three treatment groups for in vitro maturation: 120 ¼m. In Experiment 1, oocytes were pre-incubated for 1 h in TCM-199 + 20% estrous canine serum (SCE) + cysteamine + OA (0.5 ¼M). Then, oocytes were cultured for 48 h in the same medium without OA at 38.5°C, 5% CO2 in air. As a control group, oocytes were matured in vitro under the same conditions but without pre-incubation with OA. In Experiment 2, to determine if the effect of OA is mediated by cumulus cells, >120 ¼m oocytes were denuded from cumulus cells, incubated with or without OA, and cultured in vitro as previously described. At 48 h, all oocytes were stained and fixed with glycerol-Hoechst 33342 to assess the stage of meiotic maturation. In Experiment 1, OA induced a significantly higher incidence of meiotic resumption in oocytes 120 ¼m OA group (64/78, 82.0%) was similar to the >120 ¼m control group (56/72, 77.8%), but a significantly higher proportion of the oocytes pre-incubated with OA progressed to MII than did the control oocytes (40/78, 51.3% vs. 12/72, 16.7%, respectively; P 120-¼m oocytes with (7/63, 11.1%) or without OA (7/55, 12.7%) and none of them progressed to MII. In conclusion, the results of the present study indicate that treatment of fully grown (>120 ¼m) oocytes with okadaic acid at the onset of in vitro maturation can result in a higher frequency of meiotic maturation than previously reported. Also, we determined that the beneficial effect of okadaic acid was mediated by cumulus cells.

278 MEIOTIC RESUMPTION IN VITRO OF CANINE OOCYTES: COMPARATIVE METHODS

In the bitch, mechanisms responsible for the meiotic resumption and progression of the oocytes are not known. In order to better understand cellular signals involved in canine oocyte meiosis, the present study was performed to investigate the ability of bitch oocytes to resume meiosis in vitro (1) after culture with okadaic acid (OA), and (2) after cell fusion with bovine MII oocytes. For this purpose oocytes were collected from ovaries of bitches undergoing ovariectomy. Ovaries were sliced repeatedly to release oocytes; only cumulus-oocytes complexes with two or more dense layer of cumulus cells, darkly granulated cytoplasm, and >110 μm in diameter were selected for experiments. In the first experiment, oocytes were pre-incubated for different times (1, 3, 20 h) in TCM 199 + 20% FCS + 0.5 μM or 2 μM OA and thereafter cultured for 48 h in the same medium without OA. A group of oocytes was matured in TCM 199 + 20% FCS for 72 h as control. At the end of culture, oocytes were stained with glycerol-Hoechst 33342 to evaluate meiotic stage. Results indicated that incubation with 2 μM OA for 1, 3, and 20 h determined a significantly higher (P < 0.001) meiotic resumption (GVBD) of canine oocytes compared to that in the control group, but the percentage of oocytes reaching MI and MII did not increase. Similar results were obtained after culture with 0.5 μM OA for 3 and 20 h. However, meiotic progression to MI and MII was significantly improved (P < 0.05) after incubation with 0.5 μM OA for 1 h. In the second experiment canine oocytes at GV stage were fused with MII bovine oocytes matured in vitro. This experiment was designed to test whether the high activity of MPF of MII bovine oocytes was able to determine modification of GV of canine oocytes. The zonae pellucidae from both GV and MII oocytes were removed using 0.1% pronase. Pairs of oocytes (n = 37) were agglutinated in medium containing 250 mg/mL phytohemoagglutinin placed between two electrodes in 0.5% glucose fusion medium, fused with a single pulse of direct current (1.25 KV/cm for 80 ms) and cultured for 2–3 h in TCM 199 + 10% FCS. After culture fused partners (n = 36) were stained with glycerol-Hoechst 33342 and evaluated. Results indicated that the fusion of MII bovine oocytes to GV reporter canine oocytes failed to induce nuclear membrane disassembly, chromatin condensation, or modification of canine nuclei in all of the fused combinations. These data suggest that OA induces meiotic resumption of canine oocytes. However, the cell fusion results seem to indicate that increased levels of MPF are not able to determine cell cycle progression. Furthermore, incubation times and concentration of okadaic acid could be refined to optimize a system for meiotic maturation of bitch oocytes. Table 1. Meiotic progression of bitch oocytes after incubation with okadaic acid This work was supported by MIUR (ex 40%).