Fatty Acids and Metabolomic Composition of Follicular Fluid Collected from Environments Associated with Good and Poor Oocyte Competence in Goats.

In goats, embryo oocyte competence is affected by follicle size regardless the age of the females. In previous studies we have found differences in blastocyst development between oocytes coming of small (<3 mm) and large follicles (>3 mm) in prepubertal (1−2 months-old) goats. Oocyte competence and Follicular Fluid (FF) composition changes throughout follicle growth. The aim of this study was to analyze Fatty Acids (FAs) composition and metabolomic profiles of FF recovered from small and large follicles of prepubertal goats and follicles of adult goats. FAs were analyzed by chromatography and metabolites by 1H-Nuclear Magnetic Resonance (1H-NMR) Spectrometry. The results showed important differences between adult and prepubertal follicles: (a) the presence of α,ß-glucose in adult and no detection in prepubertal; (b) lactate, -N-(CH3)3 groups and inositol were higher in prepubertal (c) the percentage of Linolenic Acid, Total Saturated Fatty Acids and n-3 PUFAs were higher in adults; and (d) the percentage of Linoleic Acid, total MUFAs, PUFAs, n-6 PUFAs and n-6 PUFAs: n-3 PUFAs ratio were higher in prepubertal goats. Not significant differences were found in follicle size of prepubertal goats, despite the differences in oocyte competence for in vitro embryo production.

Effects of melatonin on oocyte developmental competence and the role of melatonin receptor 1 in juvenile goats.

Melatonin enhances in vitro embryo development in several species by improving the oocyte developmental competence during in vitro maturation (IVM). Melatonin has a wide range of actions, from scavenging reactive oxygen species (ROS) to regulating gene expression, and it can also act by way of melatonin receptors. The aim of this study was to determine the mechanism of action of melatonin during the IVM of juvenile goat oocytes and the role of the membrane receptors. Melatonin receptor 1 was immunolocalized in cumulus cells and oocytes before and after 24 hr of IVM. The effect of melatonin on oocyte developmental competence was tested in three experimental IVM groups: (a) control, (b) 10-7  M melatonin, and (c) 10-7  M melatonin +10-7  M luzindole (an inhibitor of both melatonin receptors). After IVM oocytes were assessed for ROS levels, mitochondrial activity, adenosine 5'-triphosphate (ATP) concentration and relative gene expression (ACTB, SLC1A1, SOD1, GPx1, BAX, DNMT1, GCLC and GDF9). IVM-oocytes were in vitro fertilized and cultured under conventional conditions. Blastocyst rate and quality (differential cell count) were assessed at 8 days post-fertilization. Melatonin decreased ROS levels, increased mitochondrial activity and ATP content and increased blastocyst quality compared to control group (55.8 vs. 30.4 inner cell mass ICM, p < 0.05). There was no effect on the relative gene expression due to treatment with melatonin. In conclusion, we have showed that melatonin improves oocyte developmental competence in juvenile goats by reducing ROS levels and improving mitochondrial activity.

Linoleic (LA) and linolenic (ALA) acid concentrations in follicular fluid of prepubertal goats and their effect on oocyte in vitro maturation and embryo development.

In this study we assessed the concentration of linoleic acid (LA) and linolenic acid (ALA) in follicular fluid of prepubertal goats according to follicle size (<3mm or ≥3mm) by gas chromatography and tested the addition of different LA and ALA (LA:ALA) concentration ratios (50:50, 100:50 and 200:50µM) to the IVM medium on embryo development, mitochondrial activity, ATP concentration and relative gene expression (RPL19, ribosomal protein L19; SLC2A1, facilitated glucose transporter 1; ATF4, activating transcription factor 4; GPX1, glutathione peroxidase 1; HSPA5, heat-shock protein family A 70 kDa; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DNMT1, DNA methyltransferase 1; GCLC, glutamate-cysteine ligase catalytic subunit; SOD1, superoxide dismutase 1). Oocytes were in vitro matured, fertilised or parthenogenetically activated and zygotes were cultured following conventional protocols. LA concentration ranged from 247 to 319µM and ALA concentration from 8.39 to 41.19µM without any effect of follicle size. Blastocyst production from the different groups was: control FCS (22.33%) and BSA (19.63%), treatments 50:50 (22.58%), 100:50 (21.01%) and 200:50 (9.60%). Oocytes from the 200:50 group presented higher polyspermy and mitochondrial activity compared with controls and the rest of the treatment groups. No differences were observed in ATP concentration or relative expression of the genes measured between treatment groups. In conclusion, the low number of blastocysts obtained in the 200:50 group was caused by a high number of polyspermic zygotes, which could suggest that high LA concentration impairs oocyte membranes.

Beneficial effects of melatonin on in vitro embryo production from juvenile goat oocytes.

Melatonin is a universal antioxidant that improves in vitro embryo production in several species. The aims of this study were to determine the melatonin concentration in the ovarian follicular fluid (FF) of juvenile goats and the effect of melatonin during in vitro maturation (IVM) on embryo development. The FF melatonin concentration was 0.57--1.07×10-9 M, increasing with follicular diameter. Oocytes were matured, fertilised and cultured under conventional conditions. Blastocyst development, embryo quality and levels of reactive oxygen species (ROS) and reduced glutathione were assessed. In Experiment 1 different melatonin concentrations (10-3, 10-7, 10-9, 10-11 M) were added to the IVM medium, which contained cysteamine as antioxidant, and no differences were observed. In Experiment 2, melatonin (10-7 M) was tested in the presence or absence of cysteamine (experimental groups: melatonin, cysteamine, melatonin+cysteamine, non-antioxidant). The melatonin group presented a higher blastocyst rate than the non-antioxidant group (28.9 vs 11.7%; P<0.01) and a higher total cell number than the cysteamine group (225.1 vs 129.0; P<0.05). Oocytes from the melatonin and cysteamine groups had lower ROS levels than those from the non-antioxidant group. This study shows that melatonin is an interesting tool for improving oocyte competence in juvenile goats as it increases embryo production and quality.

Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep.

The aim of this study was to test the Brilliant Cresyl Blue (BCB) stain to select prepubertal sheep oocytes for in vitro blastocyst production. Oocyte diameter, mitochondrial activity, maturation-promoting factor (MPF) activity and mRNA relative expression (RE) of genes related to metabolism (ATPase Na(+)/K(+) transporting α 1 (ATP1A1) and cytochrome c oxidase subunit 1 (COX1)) and constitutive function of the cell (cytoplasmic polyadenylation-element-binding protein (CPEB) and S100A10) were assessed. Immature oocytes were exposed to different BCB concentrations (13, 26, 39 and 52  µM) and classified according to their cytoplasm colouration as grown BCB+ (blue cytoplasm) and growing BCB- (colourless cytoplasm). Staining oocytes with 13  µM BCB during 60  min allows selection of (BCB+) the largest (123.66  µm) and most competent oocytes to develop to the blastocyst stage (21%) with a higher number of cells (69.71 ± 6.19 s.e.m.) compared with non-stained BCB- oocytes (106.82  µm, 9% and 45.91 ± 3.35 s.e.m. respectively). Mitochondrial activity, assessed by MitoTracker Orange CMTMRos probe, was significantly higher in BCB+ than in BCB- oocytes after in vitro maturation (3369 and 1565  AU respectively). MPF activity was assessed by CDC2 kinase activity assay showing significantly higher activity at metaphase II stage in BCB+ than in BCB- oocytes (1.479 ± 0.09 and 1.184 ± 0.05 optical density respectively). The genes analysed in this work, ATP1A1, COX1, CPEB and S100A 10, did not show significant effect in mRNA RE between BCB selected oocytes. In conclusion, BCB stains larger and more competent oocytes to develop to the blastocyst stage with more active mitochondria and MPF activity and higher blastocyst cell number.

Translational Diagnostics: An In-House Pipeline to Validate Genetic Variants in Children with Undiagnosed and Rare Diseases.

Diagnosis is essential for the management and treatment of patients with rare diseases. In a group of patients, the genetic study identifies variants of uncertain significance or inconsistent with the phenotype; therefore, it is urgent to develop novel strategies to reach the definitive diagnosis. Herein, we develop the in-house Translational Diagnostics Program (TDP) to validate genetic variants as part of the diagnostic process with the close collaboration of physicians, clinical scientists, and research scientists. The first 7 of 33 consecutive patients for whom exome-based tests were not diagnostic were investigated. The TDP pipeline includes four steps: (i) phenotype assessment, (ii) literature review and prediction of in silico pathogenicity, (iii) experimental functional studies, and (iv) diagnostic decision-making. Re-evaluation of the phenotype and re-analysis of the exome allowed the diagnosis in one patient. In the remaining patients, the studies included either cDNA cloning or PCR-amplified genomic DNA, or the use of patients' fibroblasts. A comparative computational analysis of confocal microscopy images and studies related to the protein function was performed. In five of these six patients, evidence of pathogenicity of the genetic variant was found, which was validated by physicians. The current research demonstrates the feasibility of the TDP to support and resolve intramural medical problems when the clinical significance of the patient variant is unknown or inconsistent with the phenotype.

Effect of crocetin added to IVM medium for prepubertal goat oocytes on blastocyst outcomes after IVF, intracytoplasmic sperm injection and parthenogenetic activation.

Crocetin is an active constituent of saffron recently used as antioxidant for embryo culture. The aim of this study was to test the effect of crocetin added in the in vitro maturation (IVM) of prepubertal goat oocytes on the embryo development after in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and parthenogenetic activation (PA). Cumulus-oocyte complexes (COCs) were released from slaughterhouse ovaries of prepubertal goats and in vitro matured in supplemented TCM 199 medium during 24 h without (control group) and with crocetin. In Experiment 1, we evaluated the effect of the IVM supplementation with 0 µM (control), 0.5 µM, 1 µM and 2 µM of crocetin on the blastocyst development after IVF. No significant differences were obtained on blastocyst formation among groups (12, 7, 10, 11%; respectively). Although the blastocyst total cell number was higher in 1 µM crocetin group (150.7 cells) compared to the control (105.5), 0.5 µM (116.2) and 2 µM (93.7) crocetin groups, no significant differences were detected. In experiment 2, we assessed the effect of 1 µM crocetin supplementation in the IVM medium on the oocyte GSH level, ROS level and mitochondrial activity. ROS was significantly higher in the control than in the crocetin group (P < 0.05), but no differences in GSH level and mitochondrial activity were observed. In experiment 3, we evaluated the effect of 1 µM crocetin on the blastocyst development of oocytes after ICSI and PA. No statistical differences were found on blastocyst rate or cell number. However, compared with control, crocetin groups led to higher cleavage (59 vs. 67%, respectively, P = 0.09) and blastocyst rates (19 vs. 12%, respectively; P = 0.12) after ICSI. Although crocetin reduced ROS levels in prepubertal goat oocytes, it did not have a significant effect on oocyte embryo developmental competence.

Evaluation of commutability of several materials for harmonization alkaline phosphatase catalytic concentration measurements.

BACKGROUND: The International Standard ISO 18153 establish that one of the requirements to assure the metrological traceability of values for catalytic concentration of enzymes is the commutability of calibrator and control materials used in the reference measurement systems. This approach was applied to verify the commutability of several commercial stabilized materials using the recently published alkaline phosphatase IFCC primary reference procedure and two routine procedures. METHODS: ALP catalytic activity was measured in 50 serum samples and 16 commercial materials, including control materials from EQAS programs, using primary reference measurement procedure and two routine measurement procedures with AMP and DEA as buffers. Calibration materials with a value assigned by reference procedure which were proved to be commutable were used to recalculate the serum values obtained by routine procedures. RESULTS: All commercial materials showed a similar behaviour to the patient specimens when AMP vs IFCC procedures were compared. For DEA vs IFCC comparison only one calibration material and two quality control materials were commutable. Recalculation of serum results with a commutable common calibrator improves the agreement between methods changing the ratio AMP vs IFCC from 1.44 to 1.04 and DEA vs IFCC from 3.02 to 1.05. CONCLUSIONS: The use of a common commutable calibration material allows harmonizing ALP measurements and made traceable patient results to reference procedure.