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A paper describing the differentiation of swine adipose-derived stem cells into osteoblasts.

Vitrification induces ultrastructural and structural damages in different oocyte structures such as membrane, zona pellucida (ZP) and cytoplasm.1 Recently Raman Microspectroscopy (RMS) has been used to assess the changes caused by vitrification/ warming of in vitro matured ovine oocytes.2 RMS is a noninvasive technique for studying the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. Aim of the present study was to investigate the structural modifications of ZP and cytoplasm of vitrified/warmed in vitro matured bovine oocytes by RMS. Abattoir derived in vitro matured oocytes (n=171) were denuded and divided in three experimental groups: control untreated oocytes (CTR), oocytes only exposed to vitrification/warming solutions (CPA) and vitrified/warmed oocytes (VITRI). Oocytes were exposed/vitrified in 20% EG + 20% of DMSO and 0.5 M sucrose and warmed into decreasing concentrations of sucrose (1.25 M-0.3 M). RMS analysis was carried out on CTR matured oocytes (after 22h) and on CPA and VITRI oocytes at different incubation times (0,1,2,3 and 4h) after exposure/warming. The analysis of the large spectral date set, acquired by RMS were performed by Principal Component Analysis (PCA). Our experimental outcomes suggest that vitrification induce a transformation of the protein secondary structure from the α-helices to the b-sheet form in the ZP, while lipids tend to assume a more ordered configuration. Both effects induce a mechanical stiffening of ZP, which could explain the reduced fertility of vitrified oocytes with respect to the untreated ones. Intriguingly, these transformations present a certain degree of reversibility, which renders vitrified oocytes more similar to untreated cells after 2h of warming at room temperature.

Although oocyte cryopreservation has great potentials in the field of reproductive technologies, it still is an open challenge in the majority of domestic animals and little is known on the biochemical transformation induced by this process in the different cellular compartments. Raman micro-spectroscopy allows the non-invasive evaluation of the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. The aim of this work was to assess the biochemical modifications of both the zona pellucida and cytoplasm of vitrified/warmed in vitro matured bovine oocytes at different post-warming times. By taking advantage of Principal Component Analysis, we were able to shed light on the biochemical transformation induced by the cryogenic treatment, also pointing out the specific role of cryoprotective agents (CPs). Our results suggest that vitrification induces a transformation of the protein secondary structure from the α-helices to the β-sheet form, while lipids tend to assume a more packed configuration in the zona pellucida. Both modifications result in a mechanical hardening of this cellular compartment, which could account for the reduced fertility rates of vitrified oocytes. Furthermore, biochemical modifications were observed at the cytoplasmic level in the protein secondary structure, with α-helices loss, suggesting cold protein denaturation. In addition, a decrease of lipid unsaturation was found in vitrified oocytes, suggesting oxidative damages. Interestingly, most modifications were not observed in oocytes exposed to CPs, suggesting that they do not severely affect the biochemical architecture of the oocyte. Nevertheless, in oocytes exposed to CPs decreased developmental competence and increased reactive oxygen species production were observed compared to the control. A more severe reduction of cleavage and blastocyst rates after in vitro fertilization was obtained from vitrified oocytes. Our experimental outcomes also suggest a certain degree of reversibility of the induced transformations, which renders vitrified oocytes more similar to untreated cells after 2 h warming.

Preliminary experiments carried out in our laboratory indicated that—by fertilizing oocytes matured in vitro for 24–32 and 48 h—the resulting blastocyst rates decreased from 22.4 % (24 h), to 14.0 % (32 h) to ‘0’ % (48 h). The aim of this study was investigate upon the variation in the incidence of chromosomal abnormalities occurring in bovine oocytes matured in vitro for prolonged periods of time, i.e. from 24 to 32 to 48 h. Abattoir-derived oocytes were matured in vitro using standard procedures, for 24–32 and 48 h. After incubation, the COCs were treated with ialuronidase (3mg/ml) to eliminate the cumulus cells, swelled in hypotonic (KCl, 0.075 M) for 5–10 min, fixed individually on microscope slides with Carnoy fixative, air dried and stained with 5 % Giemsa. Conventional karyotypes were prepared from 50 matured oocytes for each time of maturation, providing the following results: chromosomal abnormalities, including unreduced diploid metaphases, hypo-haploidy and hypo-diploidy, increased from 12 % at 24 h, to 20 % at 32 h, to 36 % at 48 h. The Chi-square test (with Yates corrections) showed significant differences (P>0.01) in the rate of chromosome abnormalities from 24 to 48 h of maturation as well as from 32 to 48 h, whereas the differences between 24 and 32 h were not significant. These results confirm previous data and provide further evidence that bovine oocytes to be used in IVF programs should not be matured in vitro longer than 24–32 h.

The female gametes are more susceptible to chromosome segregation errors during meiosis I division and therefore they are the major contributors to the embryo aneuploidies. The evaluation of aneuploidies in bovine oocytes is useful for monitoring the reproductive health of this species and FISH is the main method employed for this purpose. To date only 2–3 chromosomes were simultaneously investigated for aneuploidy detection in cattle oocytes. In this work we propose a multi-color ZOO-FISH by the simultaneous detection of six specific chromosome painting probes on secondary oocytes matured in vitro. Standard procedures were employed for 24 h in vitro oocytes maturation, whereas specific autosomal probes were prepared by microdissection and DOP-PCR using river buffalo mitosis (2n=50). Probes were labelled with spectrum-green and -orange in a second DOP-PCR. Three sequential rounds of FISH were achieved for the same slides. Each round was realized using two probes simultaneously hybridized on MII oocytes with the corresponding first polar bodies (I pb). Slides were counterstained with DAPI in antifade. Digital images were captured in gray-scale and pseudo-colored by the software. Six specific probes, painting 3 out of 5 sub-metacentric river buffalo chromosomes (BBU 1p, 1q, 3p, 3q, 4p and 4q) were sequentially hybridized on BTA secondary oocytes with the corresponding (I pb). The different colors of the probes allowed the identification of six cattle chromosomes (BTA 1, 5, 8, 19, 27 and 28) both on MII and polar bodies evidencing no abnormalities for the investigated cells, but confirming their potential use for aneuploidy detection in bovine oocytes. This result opens further opportunity of investigation for clinical cytogenetic applications also in the other species with difficult CGH karyotype.

Excess thymidine is capable of synchronizing lymphocyte cultures in vitro by acting with a feed-back mechanism during the S phase of the cell cycle (Harper 2005 Methods Mol. Biol. 296, 157-166). The possibility to synchronize the embryonic growth can be a good strategy for future epigenetic studies. The present study was undertaken to test whether excess thymidine could also synchronize in vitro development of bovine embryos. Abattoir-derived cumulus-oocyte complexes (COC) of the Agerolese breed of cattle were matured in vitro using standard procedures. After maturation, COCs were transferred in drops of 300µL of IVF-TALP (25/drop) and covered with mineral oil. Frozen sperm from a bull were selected by centrifugation on a Percoll discontinuous gradient (45 to 80%). The pellet was diluted in IVF medium and added to the COC at the concentration of 1×10(6)spermmL(-1). After 18-20h of gametes co-incubation, presumptive zygotes were denuded and cultured in SOF medium containing different concentrations of thymidine (0, 300, 600, 1200, 2400µgmL(-1), final concentrations). The day after (Day 2) presumptive zygotes were washed four times in fresh SOF, classified morphologically under a stereomicroscope as not divided (n.d.), 2 cells, 3-8 cells, and 9-32 cells and cultured in standard SOF at 39°C in a humidified mixture of 5% CO(2),7% O(2), and 88% N(2). On Day 3, the embryos were again examined for the growth stage in relation to the synchronization effects. On Day 7, the embryos were evaluated for the final growth efficiency (cleavage stage and blastocyst formation). The experiment was replicated 4 times (except the 2400µgmL(-1) condition, which was replicated 2 times because of its clear toxic effect). Data were analyzed by ANOVA test. At Day 2, there were no differences between groups whatever the concentration (n.d.-2 cells: 59.7±11.2, 52.9±26.3, 56.1±14.6, 66.0±2.8, 38.0±11.3; 3-8 cells : 40.3±11.2, 47.1±26.3, 43.9±14.6, 34.0±2.8, 59.9±9.5; respectively for 300, 600, 1200, 2400, and control), while differences at Day 3 and 7 are shown in Table 1. The dosage of 300µgmL(-1) slowed embryo development without altering the developmental rate, whereas the other dosages were somewhat toxic to the zygotes, affecting the final percentage of blastocysts.