Steroid Receptor Coregulators Can Modulate the Action of Progesterone Receptor during the Estrous Cycle in Cow Endometrium

Nuclear receptor coregulators include coactivators and corepressors which associate with the progesterone receptor (PGR) during its activation. Fluctuations in the transcription levels of their respective genes and subsequent protein production as well as in related activities for histone acetyltransferase (HAT) and histone deacetylase (HDAC) can affect PGR function and thus change the action of progesterone (P4) in bovine endometrium during the estrous cycle. Endometrial tissue on days 2–5, 6–10, 11–16, and 17–20 of the estrous cycle was used for determination of the mRNA expression levels of coactivators P300, CREB, and SRC-1 along with corepressor NCOR-2 using Real-Time PCR, with protein levels by Western blot. Coregulators cellular localizations were assessed by immunohistochemistry whereas the activities of HAT and HDAC by using EIA. The highest levels of mRNA and proteins for all of the investigated coregulators, as well as the highest levels of activity for HAT and HDAC, were detected over days 2–16 of the estrous cycle. All of the tested coregulatory proteins were localized in the nuclei of endometrial cells. This research indicates the important role of coregulators of the PGR receptor in regulating P4 activity in endometrial cells, especially during the pre-implantation period.

Nutritional restriction during the peri-conceptional period alters the myometrial transcriptome during the peri-implantation period

This study hypothesized that female peri-conceptional undernutrition evokes transcriptomic alterations in the pig myometrium during the peri-implantation period. Myometrium was collected on days 15–16 of pregnancy from pigs fed a normal- (n = 4) or restricted-diet (n = 4) from conception until day 9th of pregnancy, and the transcriptomic profiles of the tissue were compared using Porcine (V2) Expression Microarrays 4 × 44 K. In restricted diet-fed pigs, 1021 differentially expressed genes (DEGs) with fold change ≥ 1.5, P ≤ 0.05 were revealed, and 708 of them were up-regulated. Based on the count score, the top within GOs was GO cellular components “extracellular exosome”, and the top KEGG pathway was the metabolic pathway. Ten selected DEGs, i.e. hydroxysteroid (17β) dehydrogenase 8, cyclooxygenase 2, prostaglandin F receptor, progesterone receptor membrane component 1, progesterone receptor membrane component 2, annexin A2, homeobox A10, S-phase cyclin A-associated protein in the ER, SRC proto-oncogene, non-receptor tyrosine kinase, and proliferating cell nuclear antigen were conducted through qPCR to validate microarray data. In conclusion, dietary restriction during the peri-conceptional period causes alterations in the expression of genes encoding proteins involved i.a. in the endocrine activity of the myometrium, embryo-maternal interactions, and mechanisms regulating cell cycle and proliferation.

Cytochrome P450 26A1 Modulates the Polarization of Uterine Macrophages During the Peri-Implantation Period

Uterine M1/M2 macrophages activation states undergo dynamic changes throughout pregnancy, and inappropriate macrophages polarization can cause adverse pregnancy outcomes, especially during the peri-implantation period. Our previous studies have confirmed that Cytochrome P450 26A1 (CYP26A1) can affect embryo implantation by regulating uterine NK cells and DCs. The aim of this study was to investigate whether CYP26A1 regulates the polarization of uterine macrophages in early pregnancy. Here, we observed that Cyp26a1 was significantly upregulated in M1 as compared with M2 of uterine macrophages, Raw264.7 and iBMDM. Knockdown of CYP26A1 in mice uterine significantly decreased the number of embryo implantation sites and the proportion of CD45+F4/80+CD206− M1-like uterine macrophages. Primary uterine macrophages treated with anti-CYP26A1 antibody expressed significantly lower levels of M1 markers Nos2, Il1b, Il6 and Tnf-a. In CYP26A1 knockout Raw264.7 cells, the protein levels of M1 markers TNF-α, IL-6 and CD86 were significantly decreased as compared with the wild type cells. Moreover, CYP26A1 deficiency decreased the ability to produce nitric oxide and increased the phagocytosis capacity of Raw264.7 cells under M1 stimulation state. The re-introduction of CYP26A1 partially reversed the polarization levels of M1 in CYP26A1 knockout Raw264.7 cells. CYP26A1 may regulate the polarization of uterine macrophages to M1 through Stap1 and Slc7a2. In summary, these results indicate that CYP26A1 plays a significant role in macrophage polarization, and knockdown of CYP26A1 can cause insufficient M1 polarization during the peri-implantation period, which has adverse effects on blastocyst implantation.

PSV-4 Peri-estrus ovarian, uterine, and hormonal variables influence 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. Overarching hypothesis was that peri-estrus changes in ULF volume through accumulation and resorption mechanisms influence ULF composition during the estrous cycle and early pregnancy. Objectives were (1) to characterize individual temporal and spatial changes in ULF volume, endometrial and luteal vascularity, endometrial and luteal size, and progesterone (P4) concentrations during the peri-estrus period in beef heifers and, (2) associate such changes with the metabolite composition in the ULF, four days after estrus. Fourteen Bos indicus heifers 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 (estrus = d 0). Plasma P4 was measured daily. On d 4, the uterine body was sampled using a cytology brush for targeted metabolomic analysis by mass spectrometry. Multivariate analyses clustered heifers according to ovarian, uterine, and hormonal variables in clusters A (n = 5) and B (n = 8 heifers). Individual metabolite concentrations were compared between clusters A and B by univariate analysis using t-test after FDR correction. 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 non-essential AA, sum of aromatic AA, and total AA were greater in cluster A (FDR ≤ 0.05). ULF volume dynamics and associated uterine, ovarian, and hormonal variables during the peri-estrus period presented a concerted variation among heifers, which was associated with the ULF composition four days after estrus. Potential implications for embryo receptivity and reproductive outcomes are the focus of the current investigation.

Perfusate metabolomics content and tubular transporters expression during kidney graft preservation by hypothermic machine perfusion

BackgroundIschemia-related injury during the pre-implantation period impacts kidney graft outcome. Evaluating these lesions by a non-invasive approach before transplantation could help to understand the mechanisms of graft injury and identify potential biomarkers predictive of graft outcomes. This study aims to determine metabolomic content of graft perfusion fluids and its dependence on preservation time and to explore whether tubular transporters are possibly involved in the metabolomics variations observed.MethodsKidneys were stored on hypothermic perfusion machines. We evaluated the metabolomic profiles of perfusion fluids (n=35) using Liquid Chromatography coupled with tandem Mass Spectrometry and studied the transcriptional expression of tubular transporters on pre-implantation biopsies (n=26). We used univariate and multivariate analyses to assess the impact of perfusion time on these parameters and their relationship with graft outcome.ResultsSeventy-two metabolites were found in preservation fluids at the end of perfusion, of which 40% were already present in the native conservation solution. We observed an increase of 23 metabolites with longer perfusion time and a decrease for 8. The predictive model for time-dependent variation of metabolomics content showed good performance (R2= 76%, Q2= 54%, accuracy= 41%, permutations test significant). Perfusion time had no effect on the mRNA expression of transporters. We found no correlation between metabolomics and transporters expression. Neither the metabolomics profile nor the transporters expression were predictive of graft outcome.ConclusionOur results open the way for further studies, focusing on both intra- and extra-tissue metabolome, to investigate whether transporter alterations can explain the variations observed in pre-implantation period.

Characterisation of peri-implantation endometrial Treg and identification of an altered phenotype in recurrent pregnancy loss

Recurrent Pregnancy Loss (RPL) affects 2–4% of couples, and with increasing numbers of pregnancy losses the risk of miscarrying a euploid pregnancy is increased, suggesting RPL is a pathology distinct from sporadic miscarriage that is due largely to lethal embryonic aneuploidy. There are a number of conditions associated with RPL including unspecified “immune” pathologies; one of the strongest candidates for dysregulation remains T regulatory cells as depletion in the very early stages of pregnancy in mice leads to pregnancy loss. Human endometrial Treg and conventional CD4T cells were isolated during the peri-implantation period of the menstrual cycle in normal women. We identified an endometrial Treg transcriptomic signature and validated an enhanced regulatory phenotype compared to peripheral blood Treg. Parous women had an altered endometrial Treg transcriptome compared to nulliparity, indicating acquired immune memory of pregnancy within the Treg population, by comparison endometrial conventional CD4T cells were not altered. We compared primary and secondary RPL to nulliparous or parous controls respectively. Both RPL subgroups displayed differentially expressed Treg gene transcriptomes compared to controls. We found increased cell surface S1PR1 and decreased TIGIT protein expression by Treg in primary RPL, confirming the presence of altered Treg in the peri-implantation RPL endometrium.

Intracortical Microelectrode Array Unit Yield under Chronic Conditions: A Comparative Evaluation

While microelectrode arrays (MEAs) offer the promise of elucidating functional neural circuitry and serve as the basis for a cortical neuroprosthesis, the challenge of designing and demonstrating chronically reliable technology remains. Numerous studies report “chronic” data but the actual time spans and performance measures corresponding to the experimental work vary. In this study, we reviewed the experimental durations that constitute chronic studies across a range of MEA types and animal species to gain an understanding of the widespread variability in reported study duration. For rodents, which are the most commonly used animal model in chronic studies, we examined active electrode yield (AEY) for different array types as a means to contextualize the study duration variance, as well as investigate and interpret the performance of custom devices in comparison to conventional MEAs. We observed wide-spread variance within species for the chronic implantation period and an AEY that decayed linearly in rodent models that implanted commercially-available devices. These observations provide a benchmark for comparing the performance of new technologies and highlight the need for consistency in chronic MEA studies. Additionally, to fully derive performance under chronic conditions, the duration of abiotic failure modes, biological processes induced by indwelling probes, and intended application of the device are key determinants.

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 it affects 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, four days after estrus (d 0). Fourteen B. indicus heifers 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 non-essential 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 four days after estrus.