Effects of intrauterine extracellular vesicle microRNAs on embryonic gene expression in low-fertility cows.

Embryo survival and pre-implantation development depend on uterine luminal fluid, which is believed to play a role in early embryonic death and infertility in cows. Extracellular vesicles (EVs) in the uterine luminal fluid contain microRNAs (miRNAs), crucial mediators of intercellular communication. miRNAs regulate conceptus-maternal interactions and participate in embryonic development by suppressing gene expression. Therefore, we hypothesized that miRNAs in the intrauterine EVs of low-fertility cows would hinder embryonic survival and development. EVs were collected from the bovine uterine luminal fluid of both normal- and low-fertility cows 7 days post-estrus. Small RNA-sequencing analysis of miRNAs isolated from these EVs identified eight miRNAs that were highly expressed in normal-fertility cows (normal-fertility miRNAs) and eight with elevated expression in low-fertility cows (low-fertility miRNAs). These two sets of miRNAs were transfected into hatched blastocysts via lipofection. RNA-seq following lipofection with low-fertility miRNAs identified 424 differentially expressed genes (DEGs) relative to the control; in contrast, following lipofection with normal-fertility miRNAs, seven DEGs were identified. Pathway analysis of the DEGs identified following lipofection with low-fertility miRNAs revealed substantial enrichment of mitogen-activated protein kinase (MAPK) signaling. Expression of activator protein 1 (AP1) and interferon-tau (IFNT) mRNA was significantly lower in the low-fertility miRNA transfection group than in the control. IFNT is essential for maternal pregnancy recognition. Therefore, miRNAs in intrauterine EVs from low-fertility cows at 7 days post-estrus may inhibit embryo development and suppress IFNT expression by altering MAPK signaling.

Possibility of two-dimensional ordering of cryptochrome 4a from European robin.

Cryptochrome (Cry) in some species could act as a quantum senser to detect the inclination angle of geomagnetic field, the function of which attributes the magnetic sensitivity of spins of unpaired electrons in radical pair (RP) in CRY generated by blue light irradiation. However, the effect of blue light on the structure and molecular behavior of Cry has not been well investigated. We conducted the size exclusion chromatography (SEC) and small-angle X-ray scattering (SAXS) analyses to inspect the molecular structure and behavior of cryptochrome 4a (ErCry4a) from European robin, a representative magnetosensory animal. The results indicated that ErCry4a could form flat-shape oligomers. Moreover, blue light irradiation induced the contraction of the ErCry4a molecule at the monomer scale and simultaneously accelerated the two-dimensional oligomerization of ErCry4a. This oligomerization may enhance the regularity of the two-dimensional arrangement of ErCry4a molecules, providing a positive effect for detecting the inclination angle.

Observation of the Superstructure in Fe5-xGeTe2 by X-ray Fluorescence Holography.

Fe5-xGeTe2 is a two-dimensional van der Waals material that exhibits ferromagnetic order with a high Curie temperature (TC) of around room temperature. In addition to TC, two magnetic transitions occur with decreasing temperature, and a charge-ordered state is observed at low temperatures. We employed Ge Kα X-ray fluorescence holography (XFH) for Fe5-xGeTe2 to directly investigate the local structure in the charge-ordered state, i.e., the 3×3 superstructure. The Ge Kα XFH results revealed local atomic structures around the Ge atom, thus clarifying the simultaneous locations and arrangements of the Te, Fe, and Ge atoms. The atomic positions relative to the Ge atom are useful for understanding the coexistence of the ideal 1 × 1 structure and 3×3 superstructure found in the charge-ordered state.

Structure of Bi2Rh3Se2 above and below charge density wave transition determined by Bi Lα and Lγ X-ray fluorescence holography.

The local structure of a two-dimensional layered material, Bi2Rh3Se2, in which superconducting (SC) and charge-density wave (CDW) states coexist, was investigated using Bi Lα and Lγ X-ray fluorescence holography (XFH). The crystal of Bi2Rh3Se2 adopts a monoclinic lattice with a space group of C2/m (No. 12) at room temperature; however, the structure below the CDW transition (∼240 K) is still unclear because of the difficulty in analyzing single-crystal X-ray diffraction. Therefore, information on the crystal structure below 240 K is significant to fully investigate the relationship between the SC and CDW states. Precisely, the value of ß has not been definitely determined, i.e., ß âˆ¼ 90° or ∼134° is still unclear. Therefore, the crystal structure above 240 K is still under discussion. In this study, we attempted to determine the crystal structure at 300 and 200 K by comparing the atomic images reconstructed from Bi Lγ holograms with images simulated based on crystal structure models. A Bi Lα hologram was also exploited to determine suitable atomic locations by comparison with the simulated ones. The atomic image simulated with the crystal structure of Bi2Rh3S2 below the structural phase transition temperature reproduced well the experimental atomic image at 200 K. Specifically, the line profiles of the reconstructed images at 300 and 200 K, which exactly reflect the intensity of the atomic spots, clearly indicate the structural variation above and below the CDW transition temperature, i.e., the supercell structure is suggested as the atomic location for Bi2Rh3Se2 below the CDW transition temperature.

Direct Observation of Group-V Dopant Substitutional Defects in CdTe Single Crystals.

Point defect chemistry strongly affects the fundamental properties of materials and has a decisive impact on device performance. The Group-V dopant is prominent acceptor species with high hole concentration in CdTe; however, its local atomic structure is still not clear owing to difficulties in definitive measurements and discrepancies between experimental observations and theoretical models. Herein, we report on direct observation of the local structure for the As dopant in CdTe single crystals by the X-ray fluorescence holography (XFH) technique, which is a powerful tool to visualize three-dimensional atomic configurations around a specific element. The XFH result shows the As substituting on both Cd (AsCd) and Te (AsTe) sites. Although AsTe has been well known as a shallow acceptor, AsCd has not attracted much attention and been discussed so far. Our results provide new insights into point defects by expanding the experimental XFH study in combination with theoretical first-principles studies in II-VI semiconductors.

Effect of a neurokinin 3 receptor-selective agonist administration on the embryos recovered from superovulated cows.

Superovulation (SOV) treatment of cows results in unovulated follicles and inconsistent quality of the recovered embryos. It has been demonstrated that luteinizing hormone (LH) secretion is suppressed during SOV treatment of cows, which may cause insufficient follicle development and variation in the development of recovered embryos and unovulated follicles. Pulsatile gonadotropin-releasing hormone/LH secretion is controlled by the activity of kisspeptin, neurokinin B and dynorphin (KNDy) neurons in the arcuate nucleus in many mammals. As neurokinin B promotes the activity of KNDy neurons, we hypothesized that senktide, a neurokinin B receptor agonist, has the potential as a therapeutic drug to improve the ovulation rate and quality of recovered embryos in SOV-treated cows via stimulation of LH secretion. Senktide was administered intravenously (30 or 300 nmol/min) for 2 h, beginning from 72 h after the start of SOV treatment. LH secretion was examined before and after administration, and embryos were collected 7 d after estrus. Senktide administration increased LH secretion in SOV-treated cows. The ratios of code 1, code 1 and 2, and blastocyst stage embryos to recovered embryos were increased by senktide (300 nmol/min) administration. Moreover, the mRNA levels of MTCO1, COX7C, and MTATP6 were upregulated in recovered embryos of senktide (300 nmol/min)-administered animals. These results indicate that the administration of senktide to SOV-treated cows enhances LH secretion and upregulates the expression of genes involved in mitochondrial metabolism in embryos, thereby improving embryo development and embryo quality.

Development of serial X-ray fluorescence holography for radiation-sensitive protein crystals.

X-ray fluorescence holography (XFH) is a powerful atomic resolution technique capable of directly imaging the local atomic structure around atoms of a target element within a material. Although it is theoretically possible to use XFH to study the local structures of metal clusters in large protein crystals, the experiment has proven difficult to perform, especially on radiation-sensitive proteins. Here, the development of serial X-ray fluorescence holography to allow the direct recording of hologram patterns before the onset of radiation damage is reported. By combining a 2D hybrid detector and the serial data collection used in serial protein crystallography, the X-ray fluorescence hologram can be directly recorded in a fraction of the measurement time needed for conventional XFH measurements. This approach was demonstrated by obtaining the Mn Kα hologram pattern from the protein crystal Photosystem II without any X-ray-induced reduction of the Mn clusters. Furthermore, a method to interpret the fluorescence patterns as real-space projections of the atoms surrounding the Mn emitters has been developed, where the surrounding atoms produce large dark dips along the emitter-scatterer bond directions. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structures of their functional metal clusters, and for other related XFH experiments such as valence-selective XFH or time-resolved XFH.

Antiviral Activity and Resistance Profile of the Novel HIV-1 Non-Catalytic Site Integrase Inhibitor JTP-0157602.

HIV-1 integrase (IN) is an essential enzyme for viral replication. Non-catalytic site integrase inhibitors (NCINIs) are allosteric HIV-1 IN inhibitors and a potential new class of antiretrovirals. In this report, we identified a novel NCINI, JTP-0157602, with an original scaffold. JTP-0157602 exhibited potent antiviral activity against HIV-1 and showed a serum-shifted 90% effective concentration (EC90) of 138 nM, which is comparable to those of the FDA-approved IN strand transfer inhibitors (INSTIs). This compound was fully potent against a wide range of recombinant viruses with IN polymorphisms, including amino acids 124/125, a hot spot of IN polymorphisms. In addition, JTP-0157602 retained potent antiviral activity against a broad panel of recombinant viruses with INSTI-related resistance mutations, including multiple substitutions that emerged in clinical studies of INSTIs. Resistance selection experiments of JTP-0157602 led to the emergence of A128T and T174I mutations, which are located at the lens epithelium-derived growth factor/p75 binding pocket of IN. JTP-0157602 inhibited HIV-1 replication mainly during the late phase of the replication cycle, and HIV-1 virions produced by reactivation from HIV-1 latently infected Jurkat cells in the presence of JTP-0157602 were noninfectious. These results suggest that JTP-0157602 and analog compounds can be used to treat HIV-1 infectious diseases. IMPORTANCE Non-catalytic site integrase inhibitors (NCINIs) are allosteric HIV-1 integrase (IN) inhibitors that bind to the lens epithelium-derived growth factor (LEDGF)/p75 binding pocket of IN. NCINIs are expected to be a new class of anti-HIV-1 agents. In this study, we present a novel NCINI, JTP-0157602, which has potent activity against a broad range of HIV-1 strains with IN polymorphisms. Furthermore, JTP-0157602 shows strong antiviral activity against IN strand transfer inhibitor-resistant mutations, suggesting that JTP-0157602 and its analogs are potential agents for treating HIV-1 infections. Structural modeling indicated that JTP-0157602 binds to the LEDGF/p75 binding pocket of IN, and the results of in vitro resistance induction revealed the JTP-0157602 resistance mechanism of HIV-1. These data shed light on developing novel NCINIs that exhibit potent activity against HIV-1 with broad IN polymorphisms and multidrug-resistant HIV-1 variants.

Involvement of gap junctions in the increased contraction of bovine oviducts at pre-ovulation observed in vitro.

In brief: Spontaneous contraction of oviductal smooth muscle is essential for gamete transport to the fertilization site in mammals. This study sheds light on the mechanism of elevated contraction amplitude in the bovine oviductal isthmus just before ovulation. Abstract: Rhythmic contraction of the oviducts is essential for transporting gametes and embryos at peri-ovulation; however, its regulatory mechanism during the estrous cycle is unclear. Meanwhile, it is reported that ion currents regulate muscle contraction. Our study aimed to clarify the involvement of ion channels and gap junctions in regulating oviductal motility during the estrous cycle in cattle. The isthmic sections of bovine oviducts collected just after ovulation (0-4 days after ovulation), at the mid-late luteal stage (10-17 days), and at the follicular stage (1-3 days before ovulation) were used in the experiments. The frequency and amplitude of contraction of the oviductal strips in the longitudinal direction were examined using the Magnus system. The frequency was not different among the estrous stages. Conversely, the amplitude was significantly higher at the follicular stage. The blockers of voltage-dependent calcium channels, both IP3 receptor and ryanodine receptors, chloride channel, and gap junction reduced the amplitude. Additionally, mRNA and protein expression of GJA1, a component of the gap junction, in the smooth muscle tissues of the oviductal isthmus were significantly higher in the follicular stage. In addition, estradiol-17ß (E2; 1.0 ng/mL) significantly increased GJA1 mRNA expression in cultured smooth muscle tissues after 24 h and GJA1 protein expression in cultured smooth muscle cells after 48 h. These results suggest that local levels of E2 in the oviductal isthmus ipsilateral to an ovary with a dominant follicle support the increased contraction amplitude of bovine ipsilateral oviducts by elevating the gap junction expression.

IFNT stimulates lysosomal function via type I IFN signaling pathway in pregnant bovine leukocytes.

In brief: Interferon tau (IFNT) stimulates lysosomal activation via the Janus-activated kinase in peripheral blood leukocytes during pregnancy recognition. IFNT-mediated lysosomal activation could serve as a novel marker for early pregnancy in cattle. Abstract: IFNT is important in establishing pregnancy in ruminants. Secreted IFNT in the uterus induces the expression of an interferon-stimulated gene (ISG) in uterine tissues and peripheral blood leukocytes (PBLs). In our previous study, increased lysosome and lysosomal cathepsin (CTS) activity and mRNA expression were observed in PBLs of pregnant cows on day 18 of pregnancy. However, the mechanism of IFNT stimulation in PBLs is unclear. Here, we explored the IFNT-mediated lysosomal activation mechanisms in PBLs during early pregnancy in dairy cows. PBLs collected from the peripheral blood of Holstein cows on day 18 post artificial insemination, after confirmation of their pregnancy status, were used to detect the expression of lysosomal-associated membrane protein (LAMP) 1, 2, CTSB and CTSK. Expression of all genes was significantly higher in PBLs of pregnant cows than in nonpregnant cows. In vitro IFN-mediated stimulation of PBLs collected from cows that did not undergo AI significantly increased lysosomal acidification and expression of LAMP1 and 2, as well as the activities of CTSB and CTSK. Immunodetection analysis showed an increase in LAMP1 and CTSK levels in the PBLs of day 18 pregnant cows. JAK inhibitor significantly decreased lysosomal acidification, CTSK activity, LAMP1, 2, and CTSK expression in the presence of IFNT. These results suggest that IFNT regulates lysosomal function via a type 1IFN-mediated pathway in PBLs during pregnancy recognition.

Deterioration of mitochondrial biogenesis and degradation in the endometrium is a cause of subfertility in cows.

To investigate possible causes of reproductive failure, we conducted global endometrial gene expression analyses in fertile and subfertile cows. Ingenuity pathway analysis showed that RICTOR and SIRT3 are significant upstream regulators for highly expressed genes in fertile cows, and are predicted to be activated upstream regulators of normal mitochondrial respiration. Canonical pathway analysis revealed that these highly expressed genes are involved in the activation of mitochondrial oxidative phosphorylation. Therefore, in subfertile cows, the inactivation of RICTOR and SIRT3 may correlate with decreased capacity of mitochondrial respiration. Furthermore, the expression levels of most mitochondrial DNA genes and nuclear genes encoding mitochondrial proteins were higher in subfertile cows. The mitochondrial DNA copy number was significantly higher in the endometrium of subfertile cows, whereas the ATP content did not differ between fertile and subfertile cows. Quantitative reverse transcription-PCR analysis demonstrated that the expression of PGC1a, TFAM, MFN1, FIS1, and BCL2L13 were significantly lower in subfertile cows. In addition, transmission electron microscopy images showed mitochondrial swelling in the endometrial cells of the subfertile cow. These results suggest that poor-quality mitochondria accumulate in the endometrium owing to a reduced capacity for mitochondrial biogenesis, fusion, fission, and degradation in subfertile cows, and may contribute to infertility.

Immunohistochemical identification of epithelial cell types in the isthmus of bovine oviduct: Comparison with the ampulla.

The oviductal epithelium consists of ciliated and non-ciliated cells, and their numbers vary depending on the segment of the oviduct and stage of the estrous cycle. Compared with the ampulla, fewer cyclic changes in the number of the two types of cells occur in the isthmus. Recently, we have reported that the epithelium in the ampullary oviduct is composed of many types of cells during different translational/transcriptional states, and their numbers change during the estrous cycle. However, detailed information regarding the epithelial cell subtypes lining the isthmic oviductal epithelium has not yet been reported. In this study, we aimed to identify the epithelial subtypes in the isthmus of the oviduct using immunohistochemistry. Some similarities and differences were observed between the ampulla and isthmus. As observed in the ampulla, epithelial cells of the isthmus expressed either FOXJ1 (ciliogenesis marker) or PAX8 (non-ciliated cell marker). The estrous cycle affected the number of Ki67+ cells but not that of ciliated cells. A relatively high rate of Ki67+ cells (60%) was observed at 1-4 days after the ovulation. Interestingly, unlike the ampulla, Ki67+/FOXJ1+ cells (12.6 ± 1.1%) were discovered in the isthmus. Double staining for Ki67 with FOXJ1, PAX8, or Centrin-1 (a centriole marker) revealed that Centrin-1 was localized on the apical surface of some Ki67+/FOXJ1+ cells. In conclusion, some epithelial cell subtypes exist in the isthmus of the oviduct and isthmus-specific cell subtypes have been identified. These region-specific cells may provide functional and morphological differences between the ampulla and isthmus of the oviduct.

Effects of insulin-like growth factor-1 on the mRNA expression of estradiol receptors, steroidogenic enzymes, and steroid production in bovine follicles.

Insulin-like growth factor-1 (IGF-1) plays a crucial role in follicular growth and stimulates steroid hormone production in bovine follicles. Steroid hormones are synthesized through the actions of steroidogenic enzymes, specifically STAR, CYP11A1, HSD3B, and CYP19A1 in both theca cells (TCs) and granulosa cells (GCs), under the influence of gonadotropins. Particularly, estradiol 17ß (E2) assumes a central role in follicular development and selection by activating estrogen receptors ß (ESR2) in GCs. We assessed ESR2 mRNA expression in GCs of developing follicles and investigated the impact of IGF-1 on the mRNA expression of ESR2, CYP19A1, FSHR, and LHCGR, STAR, CYP11A1, and HSD17B in cultured GCs and TCs, respectively. Additionally, we assessed the influence of IGF-1 on androstenedione (A4), progesterone (P4), and testosterone (T) production in TCs. Small-sized follicles (< 6 mm) exhibited the highest levels of ESR2 mRNA expression, whereas medium-sized follicles (7-8 mm) displayed higher levels than large-sized follicles (≥ 9 mm) (P < 0.05). IGF-1 increased the mRNA expression of ESR2, CYP19A1, and FSHR in GCs of follicles of both sizes, except for FSHR mRNA in medium-sized follicles (P < 0.05). IGF-1 significantly elevated mRNA expression of LHCGR, STAR, CYP11A1, and CYP17B in TCs of small- and medium-sized follicles (P < 0.05). Moreover, IGF-1 augmented the production of A4 and P4 but had no impact on T production in TCs of small- and medium-sized follicles. Taken together, our findings indicate that IGF-1 upregulates steroidogenic enzymes and steroid hormone production, underscoring the crucial role of IGF-1 in follicle development and selection.

Hyperthermia alters interleukin-6 production in response to lipopolysaccharide via endoplasmic reticulum stress in bovine endometrial cells.

In the postpartum period, cows experience the uterine bacterial infection and develop the endometritis. To eliminate bacteria and recover from endometritis, endometrial epithelial and stromal cells secrete the cytokine and chemokine, such as interleukin 6 (IL-6), IL-8, and monocyte chemotactic protein 1 (MCP1), to recruit immune cells. Moreover, the symptom of endometritis is prolonged in summer and we have recently indicated that hyperthermia suppresses and enhances the IL-6 production in response to lipopolysaccharide (LPS) challenge in endometrial epithelial and stromal cells, respectively. However, the mechanisms for the opposite reaction of IL-6 secretion in response to LPS challenge in both types of endometrial cells under hyperthermia conditions were still unclear. To reveal these mechanisms, both types of endometrial cells were cultured with LPS under the control (38.5°C) or hyperthermia (40.5°C) conditions and comprehensively analyzed differential gene expressions of them by RNA-seq. In addition, based on these results, we examined the effect of endoplasmic reticulum (ER) stress on the IL-6 production in both types of endometrial cells cultured with LPS under hyperthermia conditions. In comprehensive analysis, hyperthermia induced the ER stress in the endometrial stromal cells but not in the endometrial epithelial cells. Actually, we confirmed that hyperthermia increased the gene expression of BiP, ATF4, and sXBP1 and protein expression of BiP and phosphorylated inositol requiring 1, ER stress marker, in the endometrial stromal cells but not in the endometrial epithelial cells. Moreover, in the endometrial stromal cells exposed to LPS, activation and inhibition of ER stress enhanced the IL-6 production under control conditions and suppressed it under hyperthermia conditions, respectively. In this study, we could uncover the one of causes for the disruption of IL-6 production in response to LPS challenge in the endometrial cells under hyperthermia conditions. This finding might be a clue for the improvement of the symptom of endometritis in cows during summer.

X-ray fluorescence holography of biological metal sites: Application to myoglobin.

X-ray fluorescence holography (XFH) is a relatively new technique capable of providing unique three-dimensional structural information around specific atoms that act as a light source in crystalline samples. So far, XFH has typically been applied to inorganic materials such as dopants in metals and semiconductors. Here, we investigate the possibility of using XFH to visualize the metal active site in sperm whale myoglobin (Mb), a monomeric oxygen storage heme protein. We demonstrate that the atomic images reconstructed from the hologram data of crystals of carbonmonoxy myoglobin (MbCO) are moderately consistent with the crystal structure, which is also determined in this study by X-ray crystallography in the near-atomic resolution, as well as simulation results. These results open up a new avenue for the application of XFH to local atomic and electronic structure imaging of metal-sites in biomolecules.

A Chinese Medicine, Tokishakuyakusan, Increases Bovine Oviductal Tonus via G Protein-Coupled Estrogen Receptor 1.

Early embryo and sperm transport through the oviductal isthmus depends on the contraction and relaxation of the smooth muscle layers. Dysfunction of the oviduct transport is considered to be one of the causes of infertility. For human infertility, Chinese medicine is used in East Asia. Although there are many clinical reports regarding Tokishakuyakusan (TSS), there is little scientific evidence that it affects infertility. In this study, we investigated the effect of TSS on bovine oviductal contraction and relaxation via the G protein-coupled estrogen receptor 1 (GPER1). We collected bovine oviductal isthmic tissues at four stages of the estrous cycle, classified based on a macroscopic observation of the ovary. The Magnus method was used to monitor longitudinal contractility (frequency, contraction force, and tonus). The effects of TSS solution, GPER1 agonist (G-1), and antagonist (G-15) on oviductal contractility were examined. The protein expression level of GPER1 in the oviductal isthmic smooth muscle of each estrous stage was assessed by Western blotting. Although TSS did not affect frequency and contraction force, the tonus was significantly increased by TSS or G-1 at all stages (p < 0.05), and the effect was especially highest at days 1-4 after ovulation. The addition of G-15 significantly suppressed the TSS-induced increase of oviductal tonus at all stages (p < 0.05). There was no significant difference in GPER1 protein expression among the estrous stages. TSS affects oviductal contractility by increasing tonus via GPER1, and it may accelerate gamete and early embryo transport by contracting the oviducts longitudinally.

Evaluation of bovine uterine gland functions in 2D and 3D culture system.

In ruminants, uterine glands play key roles in the establishment of pregnancy by secreting various factors into the uterine lumen. Although a three-dimensional (3D) culture system has been used for investigating cellular functions in vitro, the detailed functions of uterine gland have not been fully elucidated. In this study, we examined the benefits of 3D culture system to examine the innate functions of bovine uterine glands. Isolated bovine uterine glands were cultured on Matrigel (2D) or in Matrigel (3D), respectively, and the mRNA levels of secreted proteins (SERPINA14, MEP1B, APOA1, ARSA, CTGF, and SPP1) were measured in isolated and cultured uterine glands. The protein expression of estrogen receptor ß (ERß) and progesterone receptor (PR) and the establishment of apico-basal polarity were examined. In isolated uterine glands, the mRNA levels of secreted proteins changed during the estrous cycle. Although uterine glands cultured in both 2D and 3D expressed ERß and PR, progesterone did not affect SERPINA14 mRNA expression. The expression of APOA1 mRNA in 2D cultured uterine glands did not respond to estrogen and progesterone. Additionally, the mRNA levels of secreted proteins in the 3D culture system were significantly higher than those in the 2D culture system, which might be attributed to the different cellular morphology between them. The locations of ZO-1 and ß-catenin in 2D cultured uterine glands were disordered compared with 3D cultured uterine glands. These results showed that the hormonal responsiveness of secreted factor expression and cellular morphology were different between 2D and 3D cultured bovine uterine glands.

Gene-expression profile and postpartum transition of bovine endometrial side population cells†.

The mechanism of bovine endometrial regeneration after parturition remains unclear. Here, we hypothesized that bovine endometrial stem/progenitor cells participate in the postpartum regeneration of the endometrium. Flow cytometry analysis identified the presence of side population (SP) cells among endometrial stromal cells. Endometrial SP cells were shown to differentiate into osteoblasts and adipocytes. RNA-seq data showed that the gene expression pattern was different between bovine endometrial SP cells and main population cells. Gene Set Enrichment Analysis identified the enrichment of stemness genes in SP cells. Significantly (false discovery rate < 0.01) upregulated genes in SP cells contained several stem cell marker genes. Gene ontology (GO) analysis of the upregulated genes in SP cells showed enrichment of terms related to RNA metabolic process and transcription. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of upregulated genes in SP cells revealed enrichment of signaling pathways associated with maintenance and differentiation of stem/progenitor cells. The terms involved in TCA cycles were enriched in GO and KEGG pathway analysis of downregulated genes in SP cells. These results support the assumption that bovine endometrial SP cells exhibit characteristics of somatic stem/progenitor cells. The ratio of SP cells to endometrial cells was lowest on days 9-11 after parturition, which gradually increased thereafter. SP cells were shown to differentiate into epithelial cells. Collectively, these results suggest that bovine endometrial SP cells were temporarily reduced immediately after calving possibly due to their differentiation to provide new endometrial cells.

Application of Anomalous X-ray Scattering Method to Liquid Electrolytes Used in a Battery: Local Structural Analysis around a Dilute Metallic Ion.

In liquid electrolytes used for a battery, various metal complexes are formed as a result of ion-solvent and ion-ion interactions, which strongly influence the properties of the electrolyte and thus the performance of the battery. Therefore, the structural characterization of such complexes is of great importance. In this study, the anomalous X-ray scattering (AXS) technique was applied to the potassium hydroxide solution including ∼0.3 mol % zinc, which is widely used in various batteries such as the alkaline battery. In spite of the small amount of the metallic ions, we have successfully extracted a local structure around zinc after careful data analysis. The obtained pair distribution function exhibited not only the short-range correlation corresponding to the Zn-O bond within the zincate anion but also a medium-range correlation above 3.5 Å. The present study demonstrates the capability of the AXS technique to detect local structures around dilute metallic ions in liquid electrolytes, which will largely extend the applicable range of this technique, especially to the field related to batteries.

Analysis of ciliogenesis process in the bovine oviduct based on immunohistochemical classification.

The oviductal epithelium is composed of ciliated and non-ciliated cells. The proportions of these cells change during the estrous cycle. However, the mechanism underlying this cyclic change in the cell proportions remains unclear. Our previous study indicated that ciliated cells are derived from non-ciliated cells. Here, we aimed to investigate the mechanism regulating the changes in the populations of ciliated and non-ciliated cells during the estrous cycle. To this end, we examined the numbers of cells that were positive for acetylated-α-tubulin (cilia marker), Ki67 (proliferation marker), PAX8 (non-ciliated cell marker), and FOXJ1 and MYB (ciliogenesis markers) in the epithelial cells at four different estrous stages (Stage I: days 1-4 after ovulation, Stage II: days 5-10, Stage III: days 11-17, and Stage IV: days 18-20) by immunohistochemistry. The oviductal epithelial cells expressed either FOXJ1 or PAX8. All the acetylated-α-tubulin+ cells were positive for FOXJ1, although there were a few acetylated-α-tubulin-/FOXJ1+ cells. MYB was expressed in both the FOXJ1+ and PAX8+ cells, but it was not expressed in the Ki67+ cells. The numbers of Ki67+ and MYB+ cells were the highest in Stage IV, while the numbers of FOXJ1+ and acetylated-α-tubulin+ cells were the highest in the following Stage I, suggesting that ciliogenesis is associated with the estrous cycle. Thus, based on immunological classification, the oviductal epithelium contains at least seven types of cells at different translational/transcriptional states, and their number is regulated by the estrous cycle. This cyclic event might provide an optimal environment for gamete transport, fertilization, and embryonic development.