A prematuration approach to equine IVM: considering cumulus morphology, seasonality, follicle of origin, gap junction coupling and large-scale chromatin configuration in the germinal vesicle.

Several studies report that a two-step culture where mammalian oocytes are first kept under meiosis-arresting conditions (prematuration) followed by IVM is beneficial to embryo development. The most promising results were obtained by stratifying the oocyte population using morphological criteria and allocating them to different culture conditions to best meet their metabolic needs. In this study, horse oocytes were characterised to identify subpopulations that may benefit from prematuration. We investigated gap-junction (GJ) coupling, large-scale chromatin configuration and meiotic competence in compact and expanded cumulus-oocyte complexes (COCs) according to follicle size (<1, 1-2, >2cm) and season. Then we tested the effect of cilostamide-based prematuration in compact COCs collected from follicles <1 and 1-2cm in diameter on embryo development. Meiotic competence was not affected by prematuration, whereas COCs from follicles 1-2cm in diameter yielded embryos with a higher number of cells per blastocyst than oocytes that underwent direct IVM (P<0.01, unpaired Mann-Whitney test), suggesting improved developmental competence. Oocytes collected from follicles <1cm in diameter were not affected by prematuration. This study represents an extensive characterisation of the functional properties of immature horse oocytes and is the first report of the effects of cilostamide-based prematuration in horse oocyte IVM on embryo development.

Age-related modulation of angiogenesis-regulating factors in the swine meniscus.

An in-depth knowledge of the native meniscus morphology and biomechanics in its different areas is essential to develop an engineered tissue. Meniscus is characterized by a great regional variation in extracellular matrix components and in vascularization. Then, the aim of this work was to characterize the expression of factors involved in angiogenesis in different areas during meniscus maturation in pigs. The menisci were removed from the knee joints of neonatal, young and adult pigs, and they were divided into the inner, intermediate and outer areas. Vascular characterization and meniscal maturation were evaluated by immunohistochemistry and Western blot analysis. In particular, expression of the angiogenic factor Vascular Endothelial Growth Factor (VEGF) and the anti-angiogenic marker Endostatin (ENDO) was analysed, as well as the vascular endothelial cadherin (Ve-CAD). In addition, expression of Collagen II (COLL II) and SOX9 was examined, as markers of the fibro-cartilaginous differentiation. Expression of VEGF and Ve-CAD had a similar pattern in all animals, with a significant increase from the inner to the outer part of the meniscus. Pooling the zones, expression of both proteins was significantly higher in the neonatal meniscus than in young and adult menisci. Conversely, the young meniscus revealed a significantly higher expression of ENDO compared to the neonatal and adult ones. Analysis of tissue maturation markers showed an increase in COLL II and a decrease in SOX9 expression with age. These preliminary data highlight some of the changes that occur in the swine meniscus during growth, in particular the ensemble of regulatory factors involved in angiogenesis.

In vitro maturation affects chromosome segregation, spindle morphology and acetylation of lysine 16 on histone H4 in horse oocytes.

Implantation failure and genetic developmental disabilities in mammals are caused by errors in chromosome segregation originating mainly in the oocyte during meiosis I. Some conditions, like maternal ageing or in vitro maturation (IVM), increase the incidence of oocyte aneuploidy. Here oocytes from adult mares were used to investigate oocyte maturation in a monovulatory species. Experiments were conducted to compare: (1) the incidence of aneuploidy, (2) the morphology of the spindle, (3) the acetylation of lysine 16 on histone H4 (H4K16) and (4) the relative amount of histone acetyltransferase 1 (HAT1), K(lysine) acetyltransferase 8 (KAT8, also known as MYST1), histone deacetylase 1 (HDAC1) and NAD-dependent protein deacetylase sirtuin 1 (SIRT1) mRNA in metaphase II stage oocytes that were in vitro matured or collected from peri-ovulatory follicles. The frequency of aneuploidy and anomalies in spindle morphology was increased following IVM, along with a decrease in H4K16 acetylation that was in agreement with our previous observations. However, differences in the amount of the transcripts investigated were not detected. These results suggest that the degradation of transcripts encoding for histone deacetylases and acetyltransferases is not involved in the changes of H4K16 acetylation observed following IVM, while translational or post-translational mechanisms might have a role. Our study also suggests that epigenetic instabilities introduced by IVM may affect the oocyte and embryo genetic stability.

Differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production.

STUDY QUESTION: Does the gene expression profile of cumulus cells (CC) accompanying oocytes with different degrees of chromatin compaction within the germinal vesicle (GV) reflect the oocyte's quality and response in culture during in-vitro embryo production (IVP). SUMMARY ANSWER: The transcriptomic profile of the CC is related to oocyte competence, setting the stage for the development of customized pre-maturation strategies to improve IVP. WHAT IS KNOWN ALREADY: Oocytes complete the acquisition of their competence during antral follicle development. During this period, the chromatin configuration within the GV changes dynamically and is indicative of oocyte's developmental potential. The interactions between somatic and germ cells modulate chromatin morphology and function and are critical for acquisition of oocyte competence. STUDY DESIGN, SIZE, DURATION: Bovine cumulus-oocyte complexes (COC) were isolated from 0.5 to 6 mm antral follicles. Surrounding CC were separated from the oocyte and classified as GV0, GV1, GV2 and GV3 according to the degree of the oocyte's chromatin compaction. PARTICIPANTS/MATERIALS, SETTING, METHOD: RNA extracted from CC of each group was amplified and hybridized on a bovine embryo-specific 44 K Agilent slide. The CC_GV1, CC_GV2 and CC_GV3 classes were each hybridized against the CC_GV0 class, representing an early oocyte differentiation stage with poor development competence. The data were normalized and fold changes of the differentially expressed genes were determined. Microarray data were validated using quantitative RT-PCR on selected targets. Microarray data were further analyzed through: (i) between-group analysis (BGA), which classifies the samples according to their transcriptomic profiles; (ii) cluster analysis according to the expression profile of each gene; and (iii) Ingenuity Pathway Analysis (IPA) to study gene regulation patterns and predicted functions. Furthermore, CC of each GV group were cultured and apoptotic cells were assessed after 3 h by caspase analysis. Finally, based on the analysis of CC transcriptomic profiles and the relationship between morphological features of the COC and the oocyte chromatin configuration, a customized, stage-dependent oocyte pre-maturation (pre-IVM) system was used to improve oocyte developmental potential before IVM. For this, the blastocyst rate and quality were assessed after in-vitro maturation and fertilization of pre-matured oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, quantitative RT-PCR results of a subset of five selected genes were consistent with the microarray data. Clustering analysis generated 16 clusters representing the main profiles of transcription modulation. Of the 5571 significantly differentially expressed probes, the majority (25.49%) best fitted with cluster #6 (downregulation between CC_GV0 and CC_GV1 and stable low levels in successive groups). IPA identified the most relevant functions associated with each cluster. Genes included in cluster #1 were mostly related to biological processes such as 'cell cycle' and 'cell death and survival', whereas genes included in cluster #5 were mostly related to 'gene expression'. Interestingly, 'lipid metabolism' was the most significant function identified in clusters #6, #9 and #12. IPA of gene lists obtained from each contrast (i.e., CC_GV0 vs. CC_GV1; CC_GV0 vs. CC_GV2; CC_GV0 vs. CC_GV3) revealed that the main affected function in each contrast was 'cell death and survival'. Importantly, apoptosis was predicted to be inhibited in CC_GV1 and CC_GV2, but activated in CC_GV3. Caspase analysis indicated that a low percentage of CC_GV0 was prone to undergo apoptosis but apoptosis increased significantly in CC from oocytes with condensed chromatin, reaching a peak in CC_GV3 (P < 0.05). Finally, the tailored oocyte pre-maturation strategy, based on morphological features of the COC and the oocyte chromatin configuration, demonstrated that pre-IVM improved the developmental capability of oocytes at early stages of differentiation (GV1-enriched COC) but was detrimental for oocytes at more advanced stages of development (GV2 and GV3-enriched COC). LARGE SCALE DATA: The data are available through the GEO series accession number GSE79886. LIMITATIONS, REASONS FOR CAUTION: This study was conducted with bovine samples. Whether or not the results are applicable to human oocytes requests further elucidation. Embryo transfer experiments are required to determine whether the improvement in blastocyst rates in the tailored system leads to increased live birth rates. WIDER IMPLICATIONS OF THE FINDINGS: The identification of multiple non-invasive biomarkers predictive of oocyte quality can greatly strengthen the pre-IVM approach aimed to improve IVM outcomes. These results have potentially important implications in treating human infertility and in developing breeding schemes for domestic mammals. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by NSERC Strategic Network EmbryoGENE, Canada and in part by CIG-Marie Curie Actions-Reintegration Grants within the EU 7FP (n. 303640, 'Pro-Ovum'). The authors declare no potential conflict of interest.

Natriuretic peptide precursor C delays meiotic resumption and sustains gap junction-mediated communication in bovine cumulus-enclosed oocytes.

Oocyte in vitro maturation (IVM) has become a valuable technological tool for animal breeding and cloning and the treatment of human infertility because it does not require the administration of exogenous gonadotropin to obtain fertilizable oocytes. However, embryo development after IVM is lower compared to in vivo maturation, most likely because oocytes collected for IVM are heterogeneous with respect to their developmental competencies. Attempts to improve IVM outcome have relied upon either prematuration culture (PMC) or two-step maturation strategies in the hope of normalizing variations in developmental competence. Such culture systems invoke the pharmacological arrest of meiosis, in theory providing oocytes sufficient time to complete the acquisition of developmental competence after cumulus-enclosed oocytes isolation from the follicle. The present study was designed to test the efficiency of natriuretic peptide precursor C (NPPC) as a nonpharmacologic meiosis-arresting agent during IVM in a monoovulatory species. NPPC has been shown to maintain meiotic arrest in vivo and in vitro in mice and pigs; however, the use of this molecule for PMC has yet to have been explored. Toward this end, meiotic cell cycle reentry, gap-junction functionality, and chromatin configuration changes were investigated in bovine cumulus-enclosed oocytes cultured in the presence of NPPC. Moreover, oocyte developmental competence was investigated after IVM, in vitro fertilization, and embryo culture and compared to standard IVM-in vitro fertilization protocol without PMC. Our results suggest that NPPC can be used to delay meiotic resumption and increase the developmental competence of bovine oocytes when used in PMC protocols.

The adenosine salvage pathway as an alternative to mitochondrial production of ATP in maturing mammalian oocytes.

Although the oocyte is the largest cell in the body and an unavoidable phase in life, its physiology is still poorly understood, and other cell types provide little insight into its unique nature. Even basic cellular functions in the oocyte such as energy metabolism are not yet fully understood. It is known that the mitochondria of the female gamete exhibit an immature form characterized by limited energy production from glucose and oxidative phosphorylation. We show that the bovine oocyte uses alternative means to maintain ATP production during maturation, namely, the adenosine salvage pathway. Meiosis resumption is triggered by destruction of cyclic AMP by phosphodiesterases producing adenosine monophosphate that is converted into ATP by adenylate kinases and creatine kinases. Inhibition of these enzymes decreased ATP production, and addition of their substrates restored ATP production in denuded oocytes. Addition of phosphocreatine to the oocyte maturation medium influenced the phenotype of the resulting blastocysts. We propose a model in which adenylate kinases and creatine kinases act as drivers of ATP production from added AMP during oocyte maturation.

Reductions in the number of mid-sized antral follicles are associated with markers of premature ovarian senescence in dairy cows.

High-producing dairy cows are subfertile; however, the mechanisms responsible for the decreased fertility are unknown. The aim of the present study was to test the hypothesis that culled dairy cows (4-8 years old) characterised by 'Lo' ovaries (i.e. those with <10 mid-antral follicles) are affected by premature ovarian senescence. Cows in which both ovaries were 'Lo' ovaries represented 5% of the total population analysed, and exhibited reduced ovarian size (P<0.001) and increased perifollicular stroma (P<0.05) compared with age-matched controls (i.e. cows in which both ovaries had >10 mid-antral follicles; 'Hi' ovaries). The total number of follicles, including healthy and atretic primordial, primary, secondary and small antral follicles, was lower in Lo ovaries (P<0.01). Interestingly, the primordial follicle population in Lo ovaries was lower (P<0.05) than in the control. Finally, the follicular fluid of mid-antral follicles from Lo ovaries had reduced oestradiol and anti-Müllerian hormone levels (P<0.05), but increased progesterone concentrations (P<0.05). Together, these data account for the reduced fertility of cows with Lo ovaries and are in agreement with previous observations that oocytes isolated from Lo ovaries have reduced embryonic developmental competence. Cows with a specific Lo ovary condition may represent a suitable model to address the causes of low fertility in high-yielding dairy cows, as well as the condition of premature ovarian aging in single-ovulating species.

Oocytes isolated from dairy cows with reduced ovarian reserve have a high frequency of aneuploidy and alterations in the localization of progesterone receptor membrane component 1 and aurora kinase B.

Oocytes isolated from cows of reproductive age with reduced antral follicle counts (AFC) have a diminished capacity of embryonic development, which may be related to alterations in the mechanism that directs the proper segregation of chromosomes. Because we demonstrated that progesterone receptor membrane component 1 (PGRMC1) is involved in chromosome congression and metaphase II (MII) plate formation, the present study was designed to determine 1) if the decrease in oocyte developmental competence observed in dairy cows with a reduced AFC is due to a higher incidence of aneuploidy and 2) whether alterations in PGRMC1 contributes to the incidence of aneuploidy. Oocytes from ovaries with reduced AFC and age-matched controls were matured in vitro and the occurrence of aneuploidy determined as well as the mRNA level and localization of PGRMC1. Although oocytes from ovaries with reduced AFC were capable of undergoing meiosis in vitro, these oocytes showed a 3-fold increase in aneuploidy compared to oocytes isolated from control ovaries (P < 0.05). Although Pgrmc1 mRNA levels were not altered, PGRMC1 and aurora kinase B (AURKB) failed to localize to precise focal points on MII chromosomes of oocytes from ovaries with reduced AFC. Furthermore, when oocytes of control ovaries were cultured with an inhibitor of AURKB activity, their MII plate was disrupted and PGRMC1 was not properly localized to the chromosomes. These results suggest that alterations in PGRMC1 and/or AURKB localization account in part for the increased aneuploidy and low development competence of oocytes from ovaries with reduced AFC.

The effect of cilostamide on gap junction communication dynamics, chromatin remodeling, and competence acquisition in pig oocytes following parthenogenetic activation and nuclear transfer.

In the pig, the efficiency of in vitro embryo production and somatic cell nuclear transfer (SCNT) procedures remains limited. It has been suggested that prematuration treatments (pre-IVM) based on the prolongation of a patent, bidirectional crosstalk between the oocyte and the cumulus cells through gap junction mediate communication (GJC), with the maintenance of a proper level of cAMP, could improve the developmental capability of oocytes. The aim of this study was to assess: 1) dose-dependent effects of cilostamide on nuclear maturation kinetics, 2) the relationship between treatments on GJC functionality and large-scale chromatin configuration changes, and 3) the impact of treatments on developmental competence acquisition after parthenogenetic activation (PA) and SCNT. Accordingly, cumulus-oocyte complexes were collected from 3- to 6-mm antral follicles and cultured for 24 h in defined culture medium with or without 1 µM cilostamide. GJC functionality was assessed by Lucifer yellow microinjection, while chromatin configuration was evaluated by fluorescence microscopy after nuclear staining. Cilostamide administration sustained functional coupling for up to 24 h of culture and delayed meiotic resumption, as only 25.6% of cilostamide-treated oocytes reached the pro-metaphase I stage compared to the control (69.7%; P < 0.05). Moreover, progressive chromatin condensation was delayed before meiotic resumption based upon G2/M biomarker phosphoprotein epitope acquisition using immunolocalization. Importantly, cilostamide treatment under these conditions improved oocyte developmental competence, as reflected in higher blastocyst quality after both parthenogenetic activation and SCNT.

Role of gap junction-mediated communications in regulating large-scale chromatin configuration remodeling and embryonic developmental competence acquisition in fully grown bovine oocyte.

PURPOSE: This study was aimed to test the hypothesis that gap junction mediated communications (GJC) are required to allow the progressive chromatin configuration remodeling (from GV1 to GV3) process to occur in fully grown oocytes in order to gain the final step of developmental competence acquisition, and that a premature disruption of GJC can alter this process. METHODS: Bovine cumulus-oocytes complexes collected from medium antral follicles were cultured for 2, 4, 6 and 8 h in the presence of 10(-4) IU/ml of r-hFSH and with 2 mM of the non-selective PDE inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) to prevent meiotic resumption. GJC functionality and chromatin configuration were monitored during the culture period. After meiotic arrest, the developmental capability of oocytes was assessed after IVM and IVF. RESULTS: IBMX was effective in significantly sustaining GJC up to 6 h and maintaining meiotic arrest, when compared to control group. Moreover, the percentage of oocytes with less condensed chromatin (GV1) decreased within 4 h of culture, while the proportion of GV2 oocytes gradually increased up to 6 h. Interestingly, a decline in the proportion of GV2 oocytes and an increase in the proportion of GV3 oocytes were observed after 6 h of culture, when the major drop of GJC occurred. On the contrary, when GJC were uncoupled by adding 3 mM of 1-heptanol or through cumulus cells removal, chromatin condensation occurred rapidly throughout the culture period, more promptly in denuded oocytes. Moreover, the maintenance of GJC during meiotic arrest was accompanied by a significant increase of developmental competence compared to the control, as indicated by a higher percentage of hatched blastocysts and blastocyst cell number. CONCLUSIONS: Altogether, our data indicate that both paracrine and junctional mechanisms are involved in modulating large-scale chromatin structure during the final phase of oocyte differentiation.

Changes in histone H4 acetylation during in vivo versus in vitro maturation of equine oocytes.

Epigenetic modifications are established during gametogenesis and preimplantation embryonic development. Any disturbance of the normal natural environment during these critical phases could cause alterations of the epigenetic signature. Histone acetylation is an important epigenetic modification involved in the regulation of chromatin organization and gene expression. The present study was aimed to determine whether the proper establishment of post-translational histone H4 acetylation at lysine 8 (AcH4K8), 12 (AcH4K12) and 16 (AcH4K16) of equine oocytes is adversely affected during in vitro maturation (IVM) when compared with in vivo matured oocytes collected from naturally cycling mares not undergoing ovarian hyperstimulation. The acetylation patterns were investigated by means of indirect immunofluorescence staining with specific antibodies directed against the acetylated lysine residues. Our results indicate that the acetylation state of H4 is dependent on the chromatin configuration in immature germinal vesicle (GV) stage oocytes and it changes in a residue-specific manner along with the increase of chromatin condensation. In particular, the levels of AcH4K8 and AcH4K12 increased significantly, while AcH4K16 decreased significantly from the fibrillar to the condensed state of chromatin configuration within the GV. Moreover, during meiosis, K8 and K12 were substantially deacetylated without any differences between in vivo and in vitro conditions, while K16 displayed a strong acetylation in oocytes matured in vivo, and in contrast, it was markedly deacetylated following IVM. Although the functional meaning of residue-specific acetylation during oocyte differentiation and meiotic resumption needs further investigation, our results support the hypothesis that IVM conditions can adversely affect oocyte ability to regulate the epigenetic reprogramming, critical for successful meiosis and subsequent embryonic development.

A Nuclear and Cytoplasmic Characterization of Bovine Oocytes Reveals That Cysteamine Partially Rescues the Embryo Development in a Model of Low Ovarian Reserve.

Decreased oocyte quality is a major determinant of age-associated fertility decline. Similarly, individuals affected by early ovarian aging carry low-quality oocytes. Using an established bovine model of early ovarian aging, we investigated key features of 'quality' oocyte maturation, associated with the onset of egg aneuploidy and reproductive aging, such as histone modifications, mitochondria distribution and activity, reduced glutathione (GSH) content, and gap junction functionality. Bovine ovaries were classified according to the antral follicle count (AFC), and the retrieved oocytes were processed immediately or matured in vitro. We observed alterations in several cellular processes, suggesting a multifactorial etiology of the reduced oocyte quality. Furthermore, we performed a rescue experiment for one of the parameters considered. By adding cysteamine to the maturation medium, we experimentally increased the free radical scavenger ability of the 'low competence' oocytes and obtained a higher embryo development. Our findings show that adopting culture conditions that counteract the free radicals has a positive impact on the quality of 'compromised' oocytes. Specifically, cysteamine treatment seems to be a promising option for treating aging-related deficiencies in embryo development.

Perceived barriers to physical activity according to stage of change and body mass index in the west virginia wisewoman population.

PURPOSE: Low-income Appalachian women aged 40-64 years are an understudied population relative to physical activity barriers. To inform a cardiovascular risk factor intervention targeting this population through the West Virginia WISEWOMAN program, we determined at baseline participants' perceptions about the presence of 6 barriers to physical activity and respective differences according to stage of readiness for change (SOC). METHODS: Low-income women aged 40-64 who had participated in a state cancer screening program were recruited through 13 clinics. Participants were interviewed at clinics using a health risk/behavioral survey that included questions about barriers to and SOC for physical activity. Weight, height, and waist circumference were measured. PRINCIPAL FINDINGS: Baseline screening was completed by 733 women; 84% were overweight (body mass index [BMI] 25-29.9 [24.1%]) or obese (BMI > or = 30 [59.8%]). The prevalence of barriers was greatest for lack of support (52.4%) and lack of willpower (50.8%); lack of time was reported by only 30.1%. On chi(2) test, significant differences were revealed across SOC for time (p = .026), don't need more physical activity (p = .003), and willpower (p < 001); and for time (p = .006) and don't need more (p < .000) across BMI categories. Time increased from precontemplation (21.1%) to maintenance (35%). Obese participants reported time less frequently (25.7%) than participants of normal weight (40.2%). CONCLUSIONS: Lack of willpower and support-not time-seem to be the dominant physical activity barriers in the West Virginia WISEWOMAN population. Interventions targeting willpower in early SOC and in obese participants may be promising toward increasing physical activity.

Does the Harvesting Site Influence the Osteogenic Potential of Mesenchymal Stem Cells?

Total hip arthroplasty (THA) represents one of the commonest surgical procedures in the orthopedic field. Osteointegration of the implant with native bone is essential for an optimal result; thus, the quality of the patient's bone surrounding the implant (i.e., the bone stock) is crucial. However, in some cases, the bone stock is insufficient and needs to be improved with autologous grafts rich in multipotent cells (i.e., from the iliac crest, from the head of the femur, or from the subchondral bone harvested from the acetabulum) or allogenic frozen bone. It is not known if the harvesting site may influence the osteogenic potential of these cells. Thus, our aim was to characterize and compare multipotent cells collected from the bone marrow, acetabular subchondral bone, and trabecular bone on the femoral head with a focus on osteogenic differentiation. The cells from three sources had a fibroblast-like phenotype and expressed surface antigens CD73, CD90, and CD105 and are negative to CD11b, CD34, and CD45. Although all these cells could be induced to differentiate into osteoblasts, chondrocytes, and adipocytes, they displayed different differentiation potentials. In osteogenic differentiation condition, the cells from the acetabulum had the lowest accumulation of calcium deposit while the cells originated from the bone marrow and femur created a considerably increased amount of the deposit. These findings were confirmed by quantitative polymerase chain reaction (qPCR). In chondrogenic and adipogenic conditions, bone marrow cells possessed a predominant differential capacity compared with the others, illustrated by high collagen type II expression together with a cartilage-like lacuna structure and the presence of fat-specific markers, respectively. To our knowledge, this is the first study comparing and demonstrating that the progenitor cells obtained from diverse surgical sites in hip replacement procedure share common characteristics of MSC but differ about plasticity and may provide rational for clinical application in cell therapy and bone grafting. The project number L1033 is registered with ClinicalTrials.gov NCT03369457.

Development of an in vitro test battery for assessing chemical effects on bovine germ cells under the ReProTect umbrella.

Current European legislation for the registration and authorization of chemicals (REACH) will require a dramatic increase in the use of animals for reproductive toxicity testing. Since one objective of REACH is to use vertebrates only as last resort, the development and validation of alternative methods is urgently needed. For this purpose ReProTect, an integrated research project funded by the European Union, joining together 33 partners with complementary expertise in reproductive toxicology, was designed. The study presented here describes a battery of two tests developed within ReProTect. The objective of these tests is the detection of chemical effects during the processes of oocyte maturation and fertilisation in a bovine model. The corresponding toxicological endpoints are the reaching of metaphase II and the formation of the pronuclei respectively. Fifteen chemicals have been tested (Benzo[a]pyrene, Busulfan, Butylparaben, Cadmium Chloride, Carbendazim, Cycloheximide, Diethylstilbestrol, Genistein, Ionomycin, Ketoconazole, Lindane, Methylacetoacetate, Mifepristone, Nocodazole and DMSO as solvent) demonstrating high intra-laboratory reproducibility of the tests. Furthermore, the responses obtained in both tests, for several substances, had a good correlation with the available in vivo and in vitro data. These tests therefore, could predictably become part of an integrated testing strategy that combines the bovine models with additional in vitro tests, in order to predict chemical hazards on mammalian fertility.

Rationale and pre-clinical evidences for the use of autologous cartilage micrografts in cartilage repair.

BACKGROUND: The management of cartilage lesions is an open issue in clinical practice, and regenerative medicine represents a promising approach, including the use of autologous micrografts whose efficacy was already tested in different clinical settings. The aim of this study was to characterize in vitro the effect of autologous cartilage micrografts on chondrocyte viability and differentiation and perform an evaluation of their application in racehorses affected by joint diseases. MATERIALS AND METHODS: Matched human chondrocytes and micrografts were obtained from articular cartilage using Rigenera® procedure. Chondrocytes were cultured in the presence or absence of micrografts and chondrogenic medium to assess cell viability and cell differentiation. For the pre-clinical evaluation, three racehorses affected by joint diseases were treated with a suspension of autologous micrografts and PRP in arthroscopy interventions. Clinical and radiographic follow-ups were performed up to 4 months after the procedure. RESULTS: Autologous micrografts support the formation of chondrogenic micromasses thanks to their content of matrix and growth factors, such as transforming growth factor ß (TGFß) and insulin-like growth factor 1 (IGF-1). On the other hand, no significant differences were observed on the gene expression of type II collagen, aggrecan, and SOX9. Preliminary data in the treatment of racehorses are suggestive of a potential in vivo use of micrografts to treat cartilage lesions. CONCLUSION: The results reported in this study showed the role of articular micrografts in the promoting chondrocyte differentiation suggesting their potential use in the clinical practice to treat articular lesions.

Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep.

Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and significantly increases cell fusion success. On the other hand, the culture of zona-free NT embryos requires the embryos to be cultured individually or anyway separated from each other to avoid aggregation and also requires to prolong the in vitro culture up to the blastocyst stage before transfer. Blastocyst rate is equal or higher with zona-free method as compared to zona-enclosed method while survival after cryopreservation and development to term is comparable. In conclusion, our findings, together with published data, demonstrate that the zona-free system described in this paper can significantly increase the output of NT blastocysts over the conventional zona-enclosed system.

Effectiveness of a nutrition intervention with rural low-income women.

OBJECTIVES: To evaluate Cookin' Up Health, a computer-based interactive nutrition intervention. METHODS: After randomization to intervention and control groups, 262 women in rural clinics in West Virginia completed both a baseline and 3-month follow-up survey. RESULTS: Compared to the control group, the intervention group had significantly improved scores on knowledge of dietary fats, food label reading, and readiness to eat 5 fruits and vegetables a day and foods lower in fat. CONCLUSIONS: This brief interactive nutrition intervention shows potential as a strategy to begin the process of change, but follow-up may be needed for actual behavior change to occur.

The Application of Stem Cells from Different Tissues to Cartilage Repair.

The degeneration of articular cartilage represents an ongoing challenge at the clinical and basic level. Tissue engineering and regenerative medicine using stem/progenitor cells have emerged as valid alternatives to classical reparative techniques. This review offers a brief introduction and overview of the field, highlighting a number of tissue sources for stem/progenitor cell populations. Emphasis is given to recent developments in both clinical and basic sciences. The relative strengths and weaknesses of each tissue type are discussed.

Ultra-low Doses of Follicle Stimulating Hormone and Progesterone Attenuate the Severity of Polycystic Ovary Syndrome Features in a Hyperandrogenized Mouse Model.

BACKGROUND: Polycystic-ovary syndrome (PCOS) is a reproductive illness characterized by hyperandrogenism and anovulation. Using hyperandrogenized mice, it was demonstrated that the oral administration of incremental dose of follicle stimulating hormone (FSH) attenuated some of PCOS characteristics. This work aimed to study the effect of ultra-low doses of combined FSH and progesterone orally administered on PCOS murine model. Moreover, the effect of sequential kinetic activation of administered hormones was tested. METHODS: Thirty-two female mice were used as animal model (four groups of eight animals each). Mice were hyperandrogenized by injection of dehyidroepiandrosterone diluted in sesame oil. Control group received only oil. Simultaneously, each animal daily received per os an activated or a not-activated combination of FSH (0.44 pg) plus progesterone (0.44 pg) or saline solution as control. Serum testosterone, estradiol, progesterone and luteinizing hormone were analyzed as endocrine markers and a morphological study of antral follicle was conducted. Data were analyzed by one-way ANOVA, followed by multiple comparison test. The p<0.05 was considered significant. RESULTS: Dehyidroepiandrosterone treatment increased both estradiol and progesterone serum levels, besides testosterone, while reduced luteinizing hormone (p<0.05); histological examination revealed an increase of cystic follicles (p<0.05). Irrespective of activation, the combined FSH and progesterone treatments restored estradiol level (p>0.05 vs. control group) and reduced cystic signs in the follicles (p<0.05 vs. dehyidroepiandrosterone treatment). CONCLUSION: This study indicate that ultra-low doses of FSH and progesterone orally administrated can reduce the sternness of PCOS in the mouse model and open a route for the study of innovative approaches for PCOS treatment.