Peri-estrus ovarian, uterine, and hormonal variables determine the uterine luminal fluid metabolome in beef heifers.

In cattle, uterine luminal fluid (ULF) is the main source of molecules that support embryo development and survival during the peri-implantation period. Our overarching hypothesis is that peri-estrus changes in uterine function, including ULF accumulation and absorption, are uneven among individuals, and affect ULF composition and fertility. Our objectives were (1) to characterize temporal and spatial changes in ULF volume, endometrial and luteal blood perfusion, endometrial and luteal size, and circulating progesterone concentrations during the peri-estrus period in beef heifers and (2) to associate such changes with the metabolite composition in the ULF, 4 days after estrus (d 0). Fourteen Bos indicus heifer that presented a PGF2α responsive CL received 500 µg PGF2α analog i.m. and were examined daily by rectal B-mode and pulse-wave color-Doppler ultrasonography until the fifth day after estrus (d 5). The composition of the ULF was analyzed by targeted mass spectrometry on d 4. Multivariate analyses clustered heifers according to ovarian, uterine, and hormonal variables in clusters A (n = 5) and B (n = 8 heifers). Concentrations of Pro, Ala, Leu, Gly, Val, Lys, Ile, Phe, Asp, Orn, Tyr, Arg, Trp, Suc, Cit, ADMA, the sum of essential Amino Acids (AA), sum of nonessential AA, sum of aromatic AA, and total AA were greater in cluster A (FDR ≤ 0.05). ULF volume dynamics and uterine, ovarian, and hormonal variables during the peri-estrus period presented a concerted variation among heifers within clusters, which was associated with the ULF composition 4 days after estrus.

In vitro long-term culture of isolated ovine preantral follicles: Influence of ethanol on steroid production, oocyte meiotic resumption, and metabolomic profile.

Ethanol is used routinely to dilute cell culture media supplements with little or no water solubility. This study evaluates the effect of low concentration of ethanol on the follicular development, oocyte maturation, hormone production, gene expression, and metabolomics profile of spent culture medium after long-term culture of isolated ovine preantral follicles. For this, follicles were cultured for 18 days in α-Minimum Essential Medium+ alone (control treatment) or supplemented with 100 ng/mL recombinant bovine FSH (rbFSH treatment) or with 0.2%-v/v ethanol (ethanol treatment). Ethanol treatment increased the percentage of degenerated follicles and oocytes significantly, however, it showed the highest estradiol secretion. Also, the rate of meiosis resumption was higher in ethanol treatment than Control treatment. Ethanol treatment decreased the mRNA levels of B-cell lymphoma 2 (BCL2), BCL2 associated X, Aquaporin 3, Connexin 43, Inhibin Subunit Beta A, kit ligand, Heat Shock Protein (HSP A1A) significantly when compared to the Control treatment. However, mRNA levels of cytochrome P450 family 19, and FSH receptors were significantly higher in ethanol treatment than in the Control treatment. The levels of some metabolites, which are likely amino acids, lipids, an analog of Cyclic guanosine monophosphate, and a derivative of phosphoinositol phosphate metabolism, had higher relative concentrations in ethanol and rbFSH treatments than the Control treatment. In conclusion, ethanol addition augmented the follicular and oocyte degeneration rates but increased the estradiol production and the meiotic resumption. Furthermore, the follicular metabolomic profile was similar between ethanol and rbFSH treatments being both treatments; however, different from the Control treatment.

Multiple reaction monitoring (MRM)-profiling for biomarker discovery applied to human polycystic ovarian syndrome.

RATIONALE: We describe multiple reaction monitoring (MRM)-profiling, which provides accelerated discovery of discriminating molecular features, and its application to human polycystic ovary syndrome (PCOS) diagnosis. The discovery phase of the MRM-profiling seeks molecular features based on some prior knowledge of the chemical functional groups likely to be present in the sample. It does this through use of a limited number of pre-chosen and chemically specific neutral loss and/or precursor ion MS/MS scans. The output of the discovery phase is a set of precursor/product transitions. In the screening phase these MRM transitions are used to interrogate multiple samples (hence the name MRM-profiling). METHODS: MRM-profiling was applied to follicular fluid samples of 22 controls and 29 clinically diagnosed PCOS patients. Representative samples were delivered by flow injection to a triple quadrupole mass spectrometer set to perform a number of pre-chosen and chemically specific neutral loss and/or precursor ion MS/MS scans. The output of this discovery phase was a set of 1012 precursor/product transitions. In the screening phase each individual sample was interrogated for these MRM transitions. Principal component analysis (PCA) and receiver operating characteristic (ROC) curves were used for statistical analysis. RESULTS: To evaluate the method's performance, half the samples were used to build a classification model (testing set) and half were blinded (validation set). Twenty transitions were used for the classification of the blind samples, most of them (N = 19) showed lower abundances in the PCOS group and corresponded to phosphatidylethanolamine (PE) and phosphatidylserine (PS) lipids. Agreement of 73% with clinical diagnosis was found when classifying the 26 blind samples. CONCLUSIONS: MRM-profiling is a supervised method characterized by its simplicity, speed and the absence of chromatographic separation. It can be used to rapidly isolate discriminating molecules in healthy/disease conditions by tailored screening of signals associated with hundreds of molecules in complex samples.

Differences in neonatal exposure to estradiol or testosterone on ovarian function and hormonal levels.

Exposure to an excess of androgen or estrogen can induce changes in reproductive function in adult animals that resemble polycystic ovary syndrome in humans. However, considerable differences exist among several types of animal models. Little is known about the molecular features of steroidogenesis and folliculogenesis in the ovaries of rats exposed to different sex steroids as neonates. Here, we evaluated the impact of androgen and estrogen exposure on the ovaries of adult female rats during their neonatal period in the gene expression of Lhr and Cyp17a1, two key players of steroidogenesis. We also assessed hormone levels, folliculogenesis and the theca-interstitial cell population. The study was performed on the second postnatal day in thirty female Wistar rats that were sorted into the following three intervention groups: testosterone, estradiol and vehicle (control group). The animals were euthanized 90 days after birth. The main outcomes were hormone serum levels, ovary histomorphometry and gene expression of Lhr and Cyp17a1 as analyzed via quantitative real-time PCR. We found that exposure to excess testosterone in early life increased the LH and testosterone serum levels, the LH/FSH ratio, ovarian theca-interstitial area and gene expression of Lhr and Cyp17a1 in adult rats. Estrogen induced an increase in the ovarian theca-interstitial area, the secondary follicle population and gene expression of Lhr and Cyp17a1. All animals exposed to the sex steroids presented with closed vaginas. Our data suggest that testosterone resulted in more pronounced reproductive changes than did estrogen exposure. Our results might provide some insight into the role of different hormones on reproductive development and on the heterogeneity of clinical manifestations of conditions such as polycystic ovary syndrome.

Lipid fingerprinting in women with early-onset preeclampsia: a first look.

OBJECTIVES: The aim of this preliminary study was to characterize the plasma lipid profiling of women with preeclampsia. DESIGN AND METHODS: Plasma samples of 8 pregnant women with early-onset preeclampsia and 8 normal pregnant women were evaluated. Lipids were extracted from plasma using the Bligh-Dyer protocol. The extracts were subjected to MALDI-MS. Data matrix was exported for partial least squares discriminant analysis (PLS-DA) and a parameter VIP was employed to reflect the variable importance in the discriminant analysis. The major discriminant variables were selected and underwent to Mann-Whitney U test. RESULTS: A total of 1290 ions were initially identified and twelve m/z signals were highlighted as the most important lipids for the discrimination of patients with preeclampsia. The identification of these differential lipids was carried out through Lipid Database Search. CONCLUSIONS: The main classes identified were glycerophosphocholines [GP01], glycerophosphoserines [GP03], glycerophosphoglycerols [GP04], glycosyldiradylglycerols [GL05] and glycerophosphates [GP10].

Single embryo and oocyte lipid fingerprinting by mass spectrometry.

Methods used for lipid analysis in embryos and oocytes usually involve selective lipid extraction from a pool of many samples followed by chemical manipulation, separation and characterization of individual components by chromatographic techniques. Herein we report direct analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of single and intact embryos or oocytes from various species. Biological samples were simply moisturized with the matrix solution and characteristic lipid (represented by phosphatidylcholines, sphingomyelins and triacylglycerols) profiles were obtained via MALDI-MS. As representative examples, human, bovine, sheep and fish oocytes, as well as bovine and insect embryos were analyzed. MALDI-MS is shown to be capable of providing characteristic lipid profiles of gametes and embryos and also to respond to modifications due to developmental stages and in vitro culture conditions of bovine embryos. Investigation in developmental biology of the biological roles of structural and reserve lipids in embryos and oocytes should therefore benefit from these rapid MALDI-MS profiles from single and intact species.