Combination of epigenetic erasing and mechanical cues to generate human epiBlastoids from adult dermal fibroblasts.

PURPOSE: This study is to develop a new protocol that combines the use of epigenetic cues and mechanical stimuli to assemble 3D spherical structures, arbitrarily defined "epiBlastoids," whose phenotype is remarkably similar to natural embryos. METHODS: A 3-step approach is used to generate epiBlastoids. In the first step, adult dermal fibroblasts are converted into trophoblast (TR)-like cells, combining the use of 5-azacytidine, to erase the original phenotype, with an ad hoc induction protocol, to drive cells towards TR lineage. In the second step, epigenetic erasing is applied once again, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like organoids. Specifically, erased cells are encapsulated into micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, TR-like cells are co-cultured with ICM-like spheroids in the same micro-bioreactors. Subsequently, the newly generated embryoids are transferred to microwells to favor epiBlastoid formation. RESULTS: Adult dermal fibroblasts are successfully readdressed towards TR lineage. Cells subjected to epigenetic erasing and encapsulated into micro-bioreactors rearrange in 3D ICM-like structures. Co-culture of TR-like cells and ICM-like spheroids into micro-bioreactors and microwells induces the formation of single structures with uniform shape reminiscent in vivo embryos. CDX2+ cells localized in the out layer of the spheroids, while OCT4+ cells in the inner of the structures. TROP2+ cells display YAP nuclear accumulation and actively transcribed for mature TR markers, while TROP2- cells showed YAP cytoplasmic compartmentalization and expressed pluripotency-related genes. CONCLUSION: We describe the generation of epiBlastoids that may find useful application in the assisted reproduction field.

Oocyte aging: looking beyond chromosome segregation errors.

The age-associated decline in female fertility is largely ascribable to a decrease in oocyte quality. This phenomenon is multifaceted and influenced by numerous interconnected maternal and environmental factors. An increase in the rate of meiotic errors is the major cause of the decline in oocyte developmental competence. However, abnormalities in the ooplasm accumulating with age - including altered metabolism, organelle dysfunction, and aberrant gene regulation - progressively undermine oocyte quality. Stockpiling of maternal macromolecules during folliculogenesis is crucial, as oocyte competence to achieve maturation, fertilization, and the earliest phases of embryo development occur in absence of transcription. At the same time, crucial remodeling of oocyte epigenetics during oogenesis is potentially exposed to interfering factors, such as assisted reproduction technologies (ARTs) or environmental changes, whose impact may be enhanced by reproductive aging. As the effects of maternal aging on molecular mechanisms governing the function of the human oocyte remain poorly understood, studies in animal models are essential to deepen current understanding, with translational implications for human ARTs. The present mini review aims at offering an updated and consistent view of cytoplasmic alterations occurring in oocytes during aging, focusing particularly on gene and epigenetic regulation. Appreciation of these mechanisms could inspire solutions to mitigate/control the phenomenon, and thus benefit modern ARTs.

Embryo biotechnologies in sheep: Achievements and new improvements.

To date, large-scale use of multiple ovulation and embryo transfer (MOET) programmes in ovine species is limited due to unpredictable results and high costs of hormonal stimulation and treatment. Therefore, even if considered reliable, they are not fully applicable in large-scale systems. More recently, the new prospects offered by in vitro embryo production (IVEP) through collection of oocytes post-mortem or by repeated ovum pick-up from live females suggested an alternative to MOET programmes and may be more extensively used, moving from the exclusive research in the laboratory to field application. The possibility to perform oocytes recovery from juvenile lambs to obtain embryos (JIVET) offers the great advantage to significantly reduce the generation interval, speeding the rate of genetic improvement. Although in the past decades several studies implemented novel protocols to enhance embryo production in sheep, the conditions of every single stage of IVEP can significantly affect embryo yield and successful transfer into the recipients. Moreover, the recent progresses on embryo production and freezing technologies might allow wider propagation of valuable genes in small ruminants populations and may be used for constitution of flocks without risks of disease. In addition, they can give a substantial contribution in preserving endangered breeds. The new era of gene editing might offer innovative perspectives in sheep breeding, but the application of such novel techniques implies involvement of specialized operators and is limited by relatively high costs for embryo manipulation and molecular biology analysis.

The subcortical maternal complex: emerging roles and novel perspectives.

Since its recent discovery, the subcortical maternal complex (SCMC) is emerging as a maternally inherited and crucial biological structure for the initial stages of embryogenesis in mammals. Uniquely expressed in oocytes and preimplantation embryos, where it localizes to the cell subcortex, this multiprotein complex is essential for early embryo development in the mouse and is functionally conserved across mammalian species, including humans. The complex has been linked to key processes leading the transition from oocyte to embryo, including meiotic spindle formation and positioning, regulation of translation, organelle redistribution, and epigenetic reprogramming. Yet, the underlying molecular mechanisms for these diverse functions are just beginning to be understood, hindered by unresolved interplay of SCMC components and variations in early lethal phenotypes. Here we review recent advances confirming involvement of the SCMC in human infertility, revealing an unexpected relationship with offspring health. Moreover, SCMC organization is being further revealed in terms of novel components and interactions with additional cell constituents. Collectively, this evidence prompts new avenues of investigation into possible roles during the process of oogenesis and the regulation of maternal transcript turnover during the oocyte to embryo transition.

Distribution and size of lipid droplets in oocytes recovered from young lamb and adult ovine ovaries.

This study evaluated the distribution and size of lipid droplets (LDs) in oocytes recovered from young and adult ovine ovaries. Collected oocytes were categorised on the basis of their major diameter (small (SO), 70-90 µm; medium (MO), >90-110 µm; large (LO), >110-130µm) and were stained with Nile red to detect LDs. In adult and young oocytes, a diffuse pattern distribution of LDs was dominant in all classes except adult LO and young SO and LO. Larger LDs (i.e. >3µm) were mostly present in young SO and LO, whereas smaller LDs (1-3µm) were detected in the other adult and young oocyte categories.

Post-mortem recovery, in vitro maturation and fertilization of fallow deer (Dama dama, Linnaeus 1758) oocytes collected during reproductive and no reproductive season.

Habitat degradation leads to small and fragmented populations, lower genetic variability and fertility overtime. Assisted reproductive techniques represent important tools to cope with the dramatic loss of biodiversity. Fallow deer (Dama dama), beyond its high commercial value and wide distribution, may represent the most suitable model to study endangered cervids. In this study, oocytes were recovered post-mortem from fallow deer during the breeding and no breeding seasons and were in vitro matured (IVM). The ability of cryopreserved thawed sperm samples recovered by electroejaculation from four adult males was tested by in vitro fertilization of IVM oocytes. The number of oocytes collected per ovary did significantly vary across seasons from 6.2 ± 0.92 during breeding season to 10.4 ± 1.26 during no breeding season (p = .006). Oocytes collected during the breeding season showed higher in vitro fertilization rate compared to the no breeding season (p = .045). However, no embryos reached the blastocyst stage. Semen samples obtained by electroejaculation were successfully cryopreserved, although the cryopreservation process negatively affected most kinetic parameters, mainly at 2 hr post-thawing. Moreover, the percentage of rapid spermatozoa significantly decreased between fresh samples and at 2 hr post-thawing, whereas the percentage of slow spermatozoa increased across the same period (p < .05). Our study provides the logistic steps for the application of assisted reproductive techniques in fallow deer and might be of great interest for genetic resource bank planning.

Gene expression analysis of ovine prepubertal testicular tissue vitrified with a novel cryodevice (E.Vit).

PURPOSE: Testicular tissue cryopreservation prior to gonadotoxic therapies is a method to preserve fertility in children. However, the technique still requires development, especially when the tissue is immature and rather susceptible to stress derived from in vitro manipulation. This study aimed to investigate the effects of vitrification with a new cryodevice (E.Vit) on cell membrane integrity and gene expression of prepubertal testicular tissue in the ovine model. METHODS: Pieces of immature testicular tissue (1 mm3) were inserted into "E.Vit" devices and vitrified with a two-step protocol. After warming, tissues were cultured in vitro and cell membrane integrity was assessed after 0, 2, and 24 h by trypan blue exclusion test. Controls consisted of non-vitrified tissue analyzed after 0, 2, and 24 h in vitro culture (IVC). Expression of genes involved in transcriptional stress response (BAX, SOD1, CIRBP, HSP90AB1), cell proliferation (KIF11), and germ- (ZBDB16, TERT, POU5F1, KIT) and somatic- (AR, FSHR, STAR) cell specific markers was evaluated 2 and 24 h after warming. RESULTS: Post-warming trypan blue staining showed the survival of most cells, although membrane integrity immediately after warming (66.00% ± 4.73) or after 2 h IVC (59.67% ± 4.18) was significantly lower than controls (C0h 89.67% ± 1.45). Extended post-warming IVC (24 h) caused an additional decrease to 31% ± 3.46 (P < 0.05). Germ- and somatic-cell specific markers showed the survival of both cell types after cryopreservation and IVC. All genes were affected by cryopreservation and/or IVC, and moderate stress conditions were indicated by transcriptional stress response. CONCLUSIONS: Vitrification with the cryodevice E.Vit is a promising strategy to cryopreserve prepubertal testicular tissue.

Delay in maternal transcript degradation in ovine embryos derived from low competence oocytes.

Oocytes from prepubertal animals have a reduced ability to undergo embryo development and produce viable offspring. The present work used an ovine model consisting of oocytes derived from adult and prepubertal donors to assess the molecular status of oocytes and preimplantation embryos with different developmental competence. The lower potential of oocytes of young donors was confirmed in terms of in vitro developmental capabilities and kinetics. A panel of genes including maternal effect (DPPA3, GDF9, NMP2, ZAR1) and housekeeping genes (ACTB, RPL19, SDHA, YWHAZ, ATP1A1), genes involved in DNA methylation (DNMT1, DNMT3A, DNMT3B), genomic imprinting (IGF2R), pluripotency (NANOG, POU5F1) and cell cycle regulation (CCNB1, CDK1, MELK) was relatively quantified. Temporal analysis during oocyte maturation and preimplantation embryo development evidenced patterns associated with donor age. With a few gene-specific exceptions, the differential model showed a reduced transcript abundance in immature prepubertal oocytes that completely reversed trend after fertilization, when higher mRNA levels were consistently observed in early embryos, indicating a delay in maternal transcript degradation. We propose that the molecular shortage in the prepubertal oocyte may affect its developmental potential and impair the early pathways of maternal mRNA clearance in the embryo. While confirming the different potential of oocytes derived from adult and prepubertal donors, our work showed for the first time a consistent delay in maternal transcript degradation in embryos derived from low competence oocytes that interestingly recalls the delayed developmental kinetics. Such abnormal transcript persistence may hinder further development and represents a novel perspective on the complexity of developmental competence.

Testicular development in male lambs prenatally exposed to a high-starch diet.

Maternal nutrition during critical gestation periods impacts on offspring in later life; effects of high-starch maternal diet on testicular development in lambs were addressed. Dairy ewes were fed diets providing either 27% (Starch, S) or 11% (Fiber, F) of starch from mating to lambing (∼147 days; S147, F147) or for the last 75 days of gestation (S75, F75). Testes of single male lambs were measured and then sampled for histological and gene expression analyses at selected ages. Testicular dimensions and weight were similar among groups, but the total area of seminiferous tubules increased with age and tended to be higher (p = 0.057) in lambs from starch- than fiber-fed ewes. Sertoli and germ cells number increased with age, but was not influenced by maternal diet. Transcript abundances of androgen receptor (AR), insulin-like growth factor 1 (IGF1), and hydroxysteroid (17-beta) dehydrogenase 3 (HSD17B3) was similar between S147 and F147 lambs (p > 0.1). Abundance of luteinizing hormone/choriogonadotropin receptor (LHCGR) and steroidogenic acute regulatory protein (STAR) was higher in young vs older lambs, whereas insulin-like growth factor 2 (IGF2) levels increased with age. The expression of vascular endothelial growth factor A (VEGFA), Anti-Müllerian hormone (AMH), IGF1, follicle stimulating hormone receptor (FSHR), and insulin-like growth factor 2 receptor (IGF2R) was not influenced by maternal diet or lamb age (p > 0.1). In conclusion, a high-starch maternal diet did not influence gene expression, but may have affected testicular structure in infant offspring, as seen by an increase in the total area of seminiferous tubules.

Effect of exposure to CeO2 nanoparticles on ram spermatozoa during storage at 4 °C for 96 hours.

BACKGROUND: Cerium oxide nanoparticles (CeO2 NPs) are able to store and release oxygen, conferring them scavenger activity against oxidative stress. However, their effects in reproductive systems are not yet well understood. The aim of the study was to investigate the effects of exposure of refrigerated ram semen to CeO2 NPs for 96 h on the main structural and kinematic parameters of spermatozoa. METHODS: The ejaculates of 5 Sarda rams were collected, pooled and diluted in a soybean lecithin extender. Samples were exposed to increasing doses of CeO2 NPs (0, 44 and 220 µg/mL) and stored at 4 °C for 96 h. Analyses of kinematic parameters (computer assisted sperm analysis, CASA), integrity of membranes (PI/PSA staining), ROS production (H2DCFDA staining) and DNA damage (sperm chromatin structure assay with acridine orange, SCSA) were performed every 24 h (0, 24, 48, 72 and 96 h of incubation). The experiment was carried out in 6 replicates. Data were analysed by repeated measures ANOVA with Bonferroni's as post hoc test. When the assumption of normality was not met (ROS), non-parametric Kruskal-Wallis rank test was carried out. RESULTS: Exposure of ram spermatozoa to increasing doses of CeO2 NPs had a beneficial effect on the main motility parameters from 48 h of incubation onward. Velocity of sperm cells was enhanced in the groups exposed to CeO2 NPs compared to the control. Incubation with NPs had beneficial effects on the integrity of plasma membranes of spermatozoa, with higher percentage of damaged cells in the control group compared to the exposed ones. Production of ROS was not affected by exposure to NPs and its levels rose at 96 h of incubation. The integrity of DNA remained stable throughout the 96 h of storage regardless of co-incubation with NPs. CONCLUSIONS: We reported beneficial effects of CeO2 NPs on kinematic and morphologic parameters of ram semen, such as motility and membrane integrity following 96 h of exposure. Furthermore, we also proved no genotoxic effects of CeO2 NPs. These effects could not be related to an antioxidant activity of CeO2 NPs, since ROS levels in exposed cells were similar to those of unexposed ones.

Structure of preantral follicles, oxidative status and developmental competence of in vitro matured oocytes after ovary storage at 4 °C in the domestic cat model.

BACKGROUND: Storage conditions during transportation of explanted ovaries are a critical step in setting up fertility preservation protocols in both animal and human fields. Here, we evaluated the effects of ovary storage at 4 °C on the preservation of preantral follicles and oocytes retrieved from antral follicles using the domestic cat as model. METHODS: Ovaries were harvested from fifty-five healthy domestic queens during ovariectomy and stored at 4 °C for 0 (control), 24, 48, 72 and 96 h. In Experiment 1, the effects of the storage period at 4 °C on the morphology, cytoskeleton (α/ß tubulin) and DNA integrity (phosphorylation of histone H2AX) of preantral follicles were investigated. In Experiment 2, oocytes recovered from antral follicles were matured and fertilized in vitro to evaluate their meiotic and developmental competence. Reactive oxygen species (ROS), glutathione (GSH) and lipid peroxidation were measured in matured oocytes. RESULTS: The results showed that: a) storage up to 24 h did not affect the morphology and the DNA integrity of preantral follicles; b) extended storage times caused progressive morphological abnormalities, disassembling of microtubules and DNA damage; c) storage up to 48 h did not influence in vitro meiotic maturation of oocytes nor cleavage after in vitro fertilization. However, only oocytes stored within the ovary for 24 h produced blastocysts in a percentage similar to control oocytes; d) GSH levels of in vitro matured oocytes did not change at any time during ovary storage; a progressive increase in ROS levels was detected from 48 h associated with elevated lipid peroxidation at 72 and 96 h of storage. CONCLUSIONS: Storage of cat ovaries for up to 24 h caused minimal alteration of preantral follicles and oocytes. The extension of the storage period beyond 24 h progressively impaired the structure of follicles, and modified the oxidative status of in vitro matured oocytes and their developmental competence after in vitro fertilization. This information may help when setting up programs for fertility conservation, especially for wild feline species which die in geographic areas located far away from ARTs centers.

High post-thaw survival of ram sperm after partial freeze-drying.

BACKGROUND: Recrystallization damages occur when a frozen sample is held at high subzero temperatures and when the warming process is too slow. METHODS: In this work, ram semen diluted in two different concentrations of sugar solutions (Lyo A consisted of 0.4 M sorbitol and 0.25 M trehalose, and the second, Lyo B composed of 0.26 M sorbitol and 0.165 M trehalose) in egg yolk and Tris medium were compared after freezing 10 µL samples to: (1) - 10, - 25, and - 35 °C and thawing. (2) Freezing to - 10 and - 25 °C, holding for 1 h and then thawing, and (3) freezing to - 10 and - 25 °C and drying for 1 h at these temperatures at a vacuum of 80 mTorr, prior thawing. For drying, we used a new freeze-drying apparatus (Darya, FertileSafe, Israel) having a condensation temperature below - 110 °C and a vacuum pressure of 10-100 mTorr that is reached in less than 10s. RESULTS: Results showed that samples in Lyo B solution frozen at - 25 °C had significantly higher sperm motility in partially freeze-dried samples than frozen samples (46.6 ± 2.8% vs 1.2 ± 2.5%, P < 0.001). Moreover, partially dried samples in Lyo B showed higher motility than Lyo A at - 25 °C (46.6 ± 2.8% vs 35 ± 4%). Cryomicroscopy and low-temperature/low-pressure environmental scanning electronic microscope demonstrated that the amount of the ice crystals present in partially dried samples was lower than in the frozen samples. CONCLUSION: Holding the sperm at high subzero temperatures is necessary for the primary drying of cells during the freeze-drying process. Rapid freeze-drying can be achieved using this new device, which enables to reduce recrystallization damages.

Methylation dynamics during folliculogenesis and early embryo development in sheep.

Genome-wide DNA methylation reprogramming occurs during mammalian gametogenesis and early embryogenesis. Post-fertilization demethylation of paternal and maternal genomes is considered to occur by an active and passive mechanism respectively, in most mammals but sheep; in this species no loss of methylation was observed in either pronucleus. Post-fertilization reprogramming relies on methylating and demethylating enzymes and co-factors that are stored during oocyte growth, concurrently with the re-methylation of the oocyte itself. The crucial remodelling of the oocyte epigenetic baggage often overlaps with potential interfering events such as exposure to assisted reproduction technologies or environmental changes. Here, we report a temporal analysis of methylation dynamics during folliculogenesis and early embryo development in sheep. We characterized global DNA methylation and hydroxymethylation by immunofluorescence and relatively quantified the expression of the enzymes and co-factors mainly responsible for their remodelling (DNA methyltransferases (DNMTs), ten-eleven translocation (TET) proteins and methyl-CpG-binding domain (MBD) proteins). Our results illustrate for the first time the patterns of hydroxymethylation during oocyte growth. We observed different patterns of methylation and hydroxymethylation between the two parental pronuclei, suggesting that male pronucleus undergoes active demethylation also in sheep. Finally, we describe gene-specific accumulation dynamics for methylating and demethylating enzymes during oocyte growth and observe patterns of expression associated with developmental competence in a differential model of oocyte potential. Our work contributes to the understanding of the methylation dynamics during folliculogenesis and early embryo development and improves the overall picture of early rearrangements that will originate the embryo epigenome.

Evaluation of the impact of vitrification on the actin cytoskeleton of in vitro matured ovine oocytes by means of Raman microspectroscopy.

PURPOSE: Investigation of the changes induced by vitrification on the cortical F-actin of in vitro matured ovine oocytes by Raman microspectroscopy (RMS). METHODS: Cumulus-oocyte complexes, recovered from the ovaries of slaughtered sheep, were matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. The cortical region of metaphase II (MII) oocytes (1) exposed to vitrification solutions but not cryopreserved (CPA-exp), (2) vitrified/warmed (VITRI), and (3) untreated (CTR) was analyzed by RMS. A chemical map of one quadrant of single CPA-exp, VITRI and CTR oocytes was, also, performed. In order to identify the region of Raman spectra representative of the cortical F-actin modification, a group of in vitro matured oocytes were incubated with latrunculin-A (LATA), a specific F-actin destabilizing drug, and processed for RMS analysis. Thereafter, all the oocytes were stained with rhodamine phalloidin and evaluated by fluorescence confocal microscopy. Raman spectra of the oocytes were, statistically, analyzed using Principal Component Analysis (PCA). RESULTS: The PCA score plots showed a marked discrimination between CTR oocytes and CPA-exp/ VITRI groups. The main differences, highlighted by PCA loadings, were referable to proteins (1657, 1440 and 1300 cm(-1)) and, as indicated by LATA experiments, also included the changes of the F-actin. Analysis by confocal microscopy revealed a clear alteration of the cortical F-actin of CPA-exp and VITRI oocytes confirming RMS results. CONCLUSIONS: Raman microspectroscopy may represent an alternative analytical tool for investigating the biochemical modification of the oocyte cortex, including the F-actin cytoskeleton, during vitrification of in vitro matured ovine oocytes.

Expression of maternally derived KHDC3, NLRP5, OOEP and TLE6 is associated with oocyte developmental competence in the ovine species.

BACKGROUND: The sub-cortical maternal complex (SCMC), located in the subcortex of mouse oocytes and preimplantation embryos, is composed of at least four proteins encoded by maternal effect genes: OOEP, NLRP5/MATER, TLE6 and KHDC3/FILIA. The SCMC assembles during oocyte growth and was seen to be essential for murine zygote progression beyond the first embryonic cell divisions; although roles in chromatin reprogramming and embryonic genome activation were hypothesized, the full range of functions of the complex in preimplantation development remains largely unknown. RESULTS: Here we report the expression of the SCMC genes in ovine oocytes and pre-implantation embryos, describing for the first time its expression in a large mammalian species. We report sheep-specific patterns of expression and a relationship with the oocyte developmental potential in terms of delayed degradation of maternal SCMC transcripts in pre-implantation embryos derived from developmentally incompetent oocytes. In addition, by determining OOEP full length cDNA by Rapid Amplification of cDNA Ends (RACE) we identified two different transcript variants (OOEP1 and OOEP2), both expressed in oocytes and early embryos, but with different somatic tissue distributions. In silico translation showed that 140 aminoacid peptide OOEP1 shares an identity with orthologous proteins ranging from 95% with the bovine to 45% with mouse. Conversely, OOEP2 contains a premature termination codon, thus representing an alternative noncoding transcript and supporting the existence of aberrant splicing during ovine oogenesis. CONCLUSIONS: These findings confirm the existence of the SCMC in sheep and its key role for the oocyte developmental potential, deepening our understanding on the molecular differences underlying cytoplasmic vs nuclear maturation of the oocytes. Describing differences and overlaps in transcriptome composition between model organisms advance our comprehension of the diversity/uniformity between mammalian species during early embryonic development and provide information on genes that play important regulatory roles in fertility in nonmurine models, including the human.

Use of Epigenetic Cues and Mechanical Stimuli to Generate Blastocyst-Like Structures from Mammalian Skin Dermal Fibroblasts.

Mammalian embryogenesis is characterized by complex interactions between embryonic and extra-embryonic tissues that coordinate morphogenesis, coupling bio-mechanical and bio-chemical cues, to regulate gene expression and influence cell fate. Deciphering such mechanisms is essential to understand early embryogenesis, as well as to harness differentiation disorders. Currently, several early developmental events remain unclear, mainly due to ethical and technical limitations related to the use of natural embryos.Here, we describe a three-step approach to generate 3D spherical structures, arbitrarily defined "epiBlastoids," whose phenotype is remarkably similar to natural embryos. In the first step, adult dermal fibroblasts are converted into trophoblast-like cells, combining the use of 5-azacytidine, to erase the original cell phenotype, with an ad hoc induction protocol, to drive erased cells into the trophoblast lineage. In the second step, once again epigenetic erasing is applied, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like spheroids. More specifically, erased cells are encapsulated in micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, chemically induced trophoblast-like cells and ICM-like spheroids are co-cultured in the same micro-bioreactors. The newly generated embryoids are then transferred to microwells, to encourage further differentiation and favor epiBlastoid formation. The procedure here described is a novel strategy for in vitro generation of 3D spherical structures, phenotypically similar to natural embryos. The use of easily accessible dermal fibroblasts and the lack of retroviral gene transfection make this protocol a promising strategy to study early embryogenesis as well as embryo disorders.

Raman spectroscopy-based approach to detect aging-related oxidative damage in the mouse oocyte.

PURPOSE: Detection of chemical modifications induced by aging-related oxidative damage in mouse metaphase II (MII) oocytes by Raman microspectroscopy. METHODS: CD-1 mice at the age of 4-8 weeks (young mice) and 48-52 weeks (old mice), were superovulated and oocytes at metaphase II stage were recovered from oviducts. MII oocytes from young animals were divided into three groups: A) young oocytes, processed immediately after collection; B) in vitro aged oocytes, cultured in vitro for 10 h before processing; C) oxidative-stressed oocytes, exposed to 10 mM hydrogen peroxide for 2 min before processing. Oocytes from reproductively old mice were referred to as old oocytes (D). All the oocytes were analyzed by confocal Raman microspectroscopy. The spectra were statistically analyzed using Principal Component Analysis (PCA). RESULTS: PCA evidenced that spectra from young oocytes (A) were clearly distinguishable from those obtained from in vitro-aged, oxidative-damaged and old oocytes (B, C, D) and presented significant differences in the bands attributable to lipid components (C = C stretching, 1,659 cm⁻¹; CH2 bending, 1,450 cm⁻¹; CH3 deformation,1,345 cm⁻¹; OH bending, C-N stretching, 1,211 cm⁻¹) and protein components (amide I band,1,659 cm⁻¹; CH2 bending modes and CH3 deformation, 1,450 cm⁻¹; C-N and C-C stretching vibrations, 1,132 cm⁻¹; phenylalanine's vibration, 1,035 cm⁻¹) CONCLUSIONS: Raman spectroscopy is a valuable non-invasive tool for the identification of biochemical markers of oxidative damage and could represent a highly informative method of investigation to evaluate the oocyte quality.

Fetal Growth and Osteogenesis Dynamics during Early Development in the Ovine Species.

Increased knowledge of the developmental processes during gestation could provide valuable information on potential alterations in embryonic/fetal development. We examined the development of ovine conceptus between the 20th and 70th day of gestation with three convergent analyses: (1) uterus ultrasound examination and measurement (eco) of crown-rump length (CRL) and biparietal diameter (BPD) of the conceptus; (2) direct measurement (vivo) of CRL and BPD of the conceptus outside the uterus (3) osteo-cartilage dynamics during development by differential staining. No significant differences were observed between eco and vivo measurements for CRL and BPD in all examined concepti. CRL and BPD, instead, showed a significant positive linear correlation with gestational age. The study of osteogenesis dynamics has demonstrated a completely cartilaginous ovine fetus at up to 35 days of gestation. The ossification begins in the skull (40th day) and is almost complete between the 65th and the 70th of pregnancy. Our study highlighted that CRL and BPD are accurate parameters for gestational age estimation in the first part of sheep pregnancy and provides an overview of osteochondral temporal dynamics. Furthermore, tibia ossification is a valid parameter to estimate fetal age by ultrasound.

Raman microspectroscopy as a non-invasive tool to assess the vitrification-induced changes of ovine oocyte zona pellucida.

Cryopreservation-induced modifications of zona pellucida (ZP) have been explored to a lesser extent compared to other oocyte compartments. Different methods have been applied to identify ZP changes, but most of them are invasive and measure only few properties of ZP. Raman microspectroscopy (RMS) is a powerful technique for studying the molecular composition of cells but to date few studies have been performed on the oocytes using this method. The aim of the present study is to investigate the structural modifications of ZP of vitrified/warmed in vitro matured ovine oocytes by means of RMS. Cumulus-oocyte complexes were recovered from the ovaries of slaughtered adult sheep, matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. ZPs of vitrified/warmed oocytes (VITRI), were exposed to vitrification solutions but not cryopreserved (CPA-exp) and untreated oocytes (CTR) were analyzed by RMS. We focused our analysis on the ZP protein and carbohydrate components by analyzing the 1230-1300 cm(-1) amide III region and the 1020-1140 cm(-1) spectral range in RMS spectra, respectively. The spectral profiles in the ranges of proteins and carbohydrates were comparable between CTR and CPA-exp ZPs, whereas VITRI ZPs showed a significantly altered protein secondary structure characterized by an increase in ß-sheet content and a decrease in the α-helix content. A significant modification of the carbohydrate components was also observed. This study demonstrates that vitrification of ovine oocytes induces biochemical changes of ZP related to the secondary structure of proteins and carbohydrate residues. Cryoprotectants do not strongly alter the molecular composition of ZP which is affected mainly by cooling. Raman technology offers a powerful and non-invasive tool to assess molecular modifications induced by cryopreservation in oocytes.