Publications by authors named "Hanako Bai"

47 Publications

Deciphering two rounds of cell lineage segregations during bovine preimplantation development.

FASEB J 2021 10;35(10):e21904

Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.

Blastocyst formation gives rise to the inner cell mass (ICM) and trophectoderm (TE) and is followed by the differentiation of the epiblast (Epi) and primitive endoderm (PrE) within the ICM. Although these two-round cell lineage differentiations underpin proper embryogenesis in every mammal, their spatiotemporal dynamics are quite diverse among species. Here, molecular details of the blastocyst stage in cattle were dissected using an optimized in vitro culture method. Blastocyst embryos were placed on agarose gel filled with nutrient-rich media to expose embryos to both gaseous and liquid phases. Embryos derived from this "on-gel" culture were transferred to surrogate mothers on day (D) 10 after fertilization and successfully implanted. Immunofluorescent studies using on-gel-cultured embryos revealed that the proportion of TE cells expressing the pluripotent ICM marker, OCT4, which was beyond 80% on D8, was rapidly reduced after D9 and reached 0% on D9.5. This first lineage segregation process was temporally parallel with the second one, identified by the spatial separation of Epi cells expressing SOX2 and PrE cells expressing SOX17. RNA-seq comparison of TE cells from D8 in vitro fertilized embryos and D14 in vivo embryos revealed that besides drastic reduction of pluripotency-related genes, TE cells highly expressed Wnt, FGF, and VEGF signaling pathways-related genes to facilitate the functional maturation required for feto-maternal interaction. Quantitative PCR analysis of TE cells derived from on-gel culture further confirmed time-dependent increments in the expression of key TE markers. Altogether, the present study provides platforms to understand species-specific strategies for mammalian preimplantation development.
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http://dx.doi.org/10.1096/fj.202002762RRDOI Listing
October 2021

A standardized extract of Asparagus officinalis stem improves HSP70-mediated redox balance and cell functions in bovine cumulus-granulosa cells.

Sci Rep 2021 Sep 13;11(1):18175. Epub 2021 Sep 13.

Graduate School of Global Food Resources, Hokkaido University, Sapporo, Hokkaido, 060-8589, Japan.

Heat shock (HS) protein 70 (HSP70), a well-known HS-induced protein, acts as an intracellular chaperone to protect cells against stress conditions. Although HS induces HSP70 expression to confer stress resistance to cells, HS causes cell toxicity by increasing reactive oxygen species (ROS) levels. Recently, a standardized extract of Asparagus officinalis stem (EAS), produced from the byproduct of asparagus, has been shown to induce HSP70 expression without HS and regulate cellular redox balance in pheochromocytoma cells. However, the effects of EAS on reproductive cell function remain unknown. Here, we investigated the effect of EAS on HSP70 induction and oxidative redox balance in cultured bovine cumulus-granulosa (CG) cells. EAS significantly increased HSP70 expression; however, no effect was observed on HSP27 and HSP90 under non-HS conditions. EAS decreased ROS generation and DNA damage and increased glutathione (GSH) synthesis under both non-HS and HS conditions. Moreover, EAS synergistically increased HSP70 and HSF1 expression and increased progesterone levels in CG cells. Treatment with an HSP70 inhibitor significantly decreased GSH level, increased ROS level, and decreased HSF1, Nrf2, and Keap1 expression in the presence of EAS. Furthermore, EAS significantly increased progesterone synthesis. Thus, EAS improves HSP70-mediated redox balance and cell function in bovine CG cells.
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http://dx.doi.org/10.1038/s41598-021-97632-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8437968PMC
September 2021

The use of a two-step removal protocol and optimized culture conditions improve development and quality of zona free mouse embryos.

Biochem Biophys Res Commun 2021 Nov 7;577:116-123. Epub 2021 Sep 7.

Research Faculty of Agriculture, Hokkaido University, Hokkaido, 060-8589, Japan. Electronic address:

The zona pellucida (ZP) plays an important role in both the fertilization and embryonic development. For the successful handling of early stage blastomeres for differentiation analysis, the production of identical twins or quadruplets, nuclear transfer or gene introduction requires the removal of the ZP (ZPR). Although single use of either acidic Tyrode's solution or pronase are commonly used for ZPR, long-term exposure to these agents can result in the inhibition of development with the collapse of the three-dimensional blastomere structure. Here, we demonstrate the benefits of using a two-step combined ZPR method, which relies upon a customized well-of-well (cWOW) system with smaller well size, on developmental competence and the quality of the zona free (ZF) mouse embryos. We first isolated 2-cell embryos using acid Tyrode's solution and then cultured these embryos using either commercially available or cWOW, which had a smaller microwell size. The rate of blastocyst was significantly increased by use of cWOW when compared to other culture systems. Then we evaluated the use of a two-step ZPR protocol, relying on acid Tyrode's solution and proteinase K, and subsequent culture in the cWOW system. Although acid Tyrode's solution treatment alone reduced ZPR time, blastomere morphology became wrinkled, significant decrease in blastocyst rate associated with increased number of apoptotic cells and increased expression of apoptosis-related genes were observed. Using proteinase K alone increased ZPR time and significantly decreased the blastocyst rate, but did not induce an increase in apoptotic cell number or apoptosis-related gene expression. In contrast, two-step method significantly reduced ZPR time and improved blastocyst rate by increasing the total number of cells in these wells an reducing the number of apoptotic cells in these experiments. These results suggest that the two-step ZPR protocol is beneficial for reducing the toxic effects of zona removal on ZF embryo development and quality when combined with a suitable culture system.
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http://dx.doi.org/10.1016/j.bbrc.2021.09.013DOI Listing
November 2021

Mitochondrial maturation in the trophectoderm and inner cell mass regions of bovine blastocysts.

Theriogenology 2021 Nov 2;175:69-76. Epub 2021 Sep 2.

Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan. Electronic address:

Cellular differentiation induces various morphological changes, including elongation, in mitochondria. Preimplantation embryos have round-shaped mitochondria, characteristic of undifferentiated cells. However, there is controversy regarding the precise mitochondrial morphology in blastocyst embryos, which are generated from two cell lineages: undifferentiated inner cell mass (ICM) and differentiated trophectoderm (TE). This study attempted to precisely determine mitochondrial morphology in these two blastocyst regions. Transmission electron microscopy analyses were conducted using more than 1000 mitochondria from blastocyst embryos. No significant differences were observed in the configuration of mitochondrial cristae and frequencies of hooded mitochondria, which are specific to embryos of livestock animals, between the ICM and TE. To accurately compare mitochondrial roundness between the ICM and TE, oblateness was calculated based on both the major and minor axes. Average oblateness was significantly greater in the TE than in the ICM (P < 0.01). These results indicate tissue-specific mitochondrial maturation with complete elongation in the TE at the blastocyst stage. Since mitochondrial elongation is closely associated with cellular metabolism and differentiation, the present study provides new insights for better understanding of early embryonic development in cattle.
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http://dx.doi.org/10.1016/j.theriogenology.2021.08.038DOI Listing
November 2021

Heat stress induces oxidative stress and activates the KEAP1-NFE2L2-ARE pathway in bovine endometrial epithelial cells.

Biol Reprod 2021 Jul 22. Epub 2021 Jul 22.

Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, Kita-ku Kita 9 Nishi 9, Sapporo 060-8589, Japan.

Heat stress adversely affects the reproductive function in cows. Although a relationship between heat stress and oxidative stress has been suggested, it has not been sufficiently verified in bovine endometrial epithelial cells. Here, we investigated whether oxidative stress is induced by heat stress in bovine endometrial epithelial cells under high temperature. Luciferase reporter assays showed that the reporter activity of heat shock element (HSE) and antioxidant responsive element (ARE) was increased in endometrial epithelial cells cultured under high temperature compared to that in cells cultured under basal (thermoneutral) temperature. Also, nuclear factor, erythroid 2 like 2 (NFE2L2), a master regulator of cellular environmental stress response, stabilized and the expression levels of antioxidant enzyme genes increased under high temperature. Immunostaining confirmed the nuclear localization of NFE2L2 in endometrial epithelial cells cultured under high temperature. Quantitative polymerase chain reaction analysis showed that the expression levels of representative inflammatory cytokine genes, such as prostaglandin-endoperoxide synthase 2 (PTGS2) and interleukin 8, were significantly decreased in endometrial epithelial cells cultured under high temperature compared to those in cells cultured under basal temperature. Thus, our results suggest that heat stress induces oxidative stress, whereas NFE2L2 plays a protective role in bovine endometrial epithelial cells cultured under heat stress conditions.
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http://dx.doi.org/10.1093/biolre/ioab143DOI Listing
July 2021

Loop-mediated isothermal amplification (LAMP) and machine learning application for early pregnancy detection using bovine vaginal mucosal membrane.

Biochem Biophys Res Commun 2021 Sep 10;569:179-186. Epub 2021 Jul 10.

Graduate School of Global Food Resources/Global Center for Food, Land and Water Resources, Hokkaido University, Sapporo, Hokkaido, 060-8589, Japan. Electronic address:

An early and accurate pregnancy diagnosis method is required to improve the reproductive performance of cows. Here we developed an easy pregnancy detection method using vaginal mucosal membrane (VMM) with application of Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) and machine learning. Cows underwent artificial insemination (AI) on day 0, followed by VMM-collection on day 17-18, and pregnancy diagnosis by ultrasonography on day 30. By RNA sequencing of VMM samples, three candidate genes for pregnancy markers (ISG15 and IFIT1: up-regulated, MUC16: down-regulated) were selected. Using these genes, we performed RT-LAMP and calculated the rise-up time (RUT), the first-time absorbance exceeded 0.05 in the reaction. We next determined the cutoff value and calculated accuracy, sensitivity, specificity, positive prediction value (PPV), and negative prediction value (NPV) for each marker evaluation. The IFIT1 scored the best performance at 92.5% sensitivity, but specificity was 77.5%, suggesting that it is difficult to eliminate false positives. We then developed a machine learning model trained with RUT of each marker combination to predict pregnancy. The model created with the RUT of IFIT1 and MUC16 combination showed high specificity (86.7%) and sensitivity (93.3%), which were higher compared to IFIT1 alone. In conclusion, using VMM with RT-LAMP and machine learning algorithm can be used for early pregnancy detection before the return of first estrus.
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http://dx.doi.org/10.1016/j.bbrc.2021.07.015DOI Listing
September 2021

Requirement for expression of WW domain containing transcription regulator 1 in bovine trophectoderm development.

Biochem Biophys Res Commun 2021 05 1;555:140-146. Epub 2021 Apr 1.

Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, Kita-ku, Kita 9, Nishi 9, Sapporo, 060-8589, Japan. Electronic address:

WW domain-containing transcription regulator 1 (WWTR1) is one of the primary effectors in the Hippo pathway, which plays essential roles in cell differentiation into trophectoderm (TE) and inner cell mass cell lineages at the blastocyst stage. However, little is known about the roles of WWTR1 in preimplantation development. The present study aimed to explore the significance of WWTR1 expression in preimplantation development using an mRNA knockdown (KD) system in bovine embryos. We first quantitated WWTR1 expression at protein and mRNA levels from fertilization to blastocyst stage. WWTR1 proteins gradually shifted from extranuclear localization during the 16-cell stage to nuclear localization by morula stage. WWTR1 mRNA expression was also transiently upregulated at the 16-cell stage. WWTR1 KD efficiently repressed WWTR1 expression at protein and mRNA levels. The WWTR1 KD embryos developed to the blastocyst stage at rates equivalent to those of controls, but TE cell numbers were significantly decreased. Representative TE-expressed genes, including CDX2 and IFNT were also significantly decreased in WWTR1 KD blastocysts. These results provide the first demonstration that WWTR1 expression is responsible for normal TE cell development in preimplantation embryos.
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http://dx.doi.org/10.1016/j.bbrc.2021.03.112DOI Listing
May 2021

Effect of summer heat stress on gene expression in bovine uterine endometrial tissues.

Anim Sci J 2020 Jan;91(1):e13474

Laboratory of Animal Breeding and Reproduction, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.

Heat stress negatively affects reproductive functions in cows. Increased temperature disturbs fetal development in utero. However, the effect of heat stress on uterine endometrial tissues has not been fully examined. Using qPCR analysis, we measured the mRNA expression of various molecular markers in uterine endometrial tissue of dairy cows from Hokkaido, Japan, in winter and summer. Markers examined were heat shock proteins (HSPs), antioxidant enzymes (catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, and glutathione peroxidase 4), inflammatory cytokines, and interferon stimulated genes. Our results showed heat stress, body and milk temperatures were higher during summer than during winter. Expression levels of HSP27, HSP60, and HSP90 mRNA, and of catalase and copper/zinc superoxide dismutase mRNA were lower in summer than in winter. Tumor necrosis factor alpha expression was higher in summer than in winter. In conclusion, summer heat stress may reduce the expression of HSPs, affecting the levels of inflammatory cytokines in bovine uterine endometrial tissue.
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http://dx.doi.org/10.1111/asj.13474DOI Listing
January 2020

Yes-associated protein 1 translocation through actin cytoskeleton organization in trophectoderm cells.

Dev Biol 2020 12 15;468(1-2):14-25. Epub 2020 Sep 15.

Laboratory of Animal Breeding and Reproduction, Research Faculty of Agriculture, Hokkaido University, Kita-ku Kita 9 Nishi 9, Sapporo, 060-8589, Japan. Electronic address:

A mammalian embryo experiences the first cell segregation at the blastocyst stage, in which cells giving form to the embryo are sorted into two lineages; trophectoderm (TE) and inner cell mass (ICM). This first cell segregation process is governed by cell position-dependent Hippo signaling, which is a phosphorylation cascade determining whether Yes-associated protein 1 (YAP1), one of the key components of the Hippo signaling pathway, localizes within the nucleus or cytoplasm. YAP1 localization determines the transcriptional on/off switch of a key gene, Cdx2, required for TE differentiation. However, the control mechanisms involved in YAP1 nucleocytoplasmic shuttling post blastocyst formation remain unknown. This study focused on the mechanisms involved in YAP1 release from TE nuclei after blastocoel contraction in bovine blastocysts. The blastocysts contracted by blastocoel fluid aspiration showed that the YAP1 translocation from nucleus to cytoplasm in the TE cells was concomitant with the protruded actin cytoskeleton. This YAP1 release from TE nuclei in the contracted blastocysts was prevented by actin disruption and stabilization. In contrast, Y27632, which is a potent inhibitor of Rho-associated coiled-coil containing protein kinase 1/2 (ROCK) activity, was found to promote YAP1 nuclear localization in the TE cells of contracted blastocysts. Meanwhile, lambda protein phosphatase (LPP) treatment inducing protein dephosphorylation could not prevent YAP1 release from TE nuclei in the contracted blastocysts, indicating that YAP1 release from TE nuclei does not depend on the Hippo signaling pathway. These results suggested that blastocyst contraction causes YAP1 release from TE nuclei through actin cytoskeleton remodeling in a Hippo signaling-independent manner. Thus, the present study raised the possibility that YAP1 subcellular localization is controlled by actin cytoskeletal organization after the blastocyst formation. Our results demonstrate diverse regulatory mechanisms for YAP1 nucleocytoplasmic shuttling in TE cells.
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http://dx.doi.org/10.1016/j.ydbio.2020.09.004DOI Listing
December 2020

Cluster analysis to evaluate disease risk in periparturient dairy cattle.

Anim Sci J 2020 Jan;91(1):e13442

Doctoral Program in Advanced Agricultural Technology & Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki Prefecture, Japan.

Predicting periparturient disease risk is of immense value to the dairy industry. Periparturient diseases are interrelated with each other; however, predicting the onset risk of these diseases has predominantly been based on a single blood parameter for a single disease. This study examined a new diagnostic method to predict the risk of periparturient diseases. We conducted cluster analysis of multiple blood constituents from 20 Holstein cattle at 1 week post-partum, and the cattle were divided into two groups, A or B. We then compared the periparturient and early-lactation blood constituents of these groups. Group B had significantly higher 3-hydroxybutyric acid concentrations and were suspected to have subclinical ketosis. Group B also had significantly lower calcium concentrations, with a tendency for subclinical hypocalcemia. We also performed discriminant analysis using blood parameters at 1 week post-partum, which grouped the population into the same two groups as the cluster analysis based on three variables: inorganic phosphorus, calcium, and either phospholipids or total cholesterol. We further showed that these discriminant functions could be used to predict the risk of periparturient disease even before parturition. Our results indicate that cluster analysis with multiple blood constituents is useful for predicting periparturient disease risks.
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http://dx.doi.org/10.1111/asj.13442DOI Listing
January 2020

The role of RHOA signaling in trophectoderm cell-fate decision in cattle.

Biochem Biophys Res Commun 2020 08 6;528(4):713-718. Epub 2020 Jun 6.

Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, Kita-ku, Kita 9, Nishi 9, Sapporo, 060-8589, Japan. Electronic address:

Mammalian blastocysts are composed of two distinct cell lineages, namely the inner cell mass (ICM) and trophectoderm (TE). TE cells that give rise to the embryonic placenta are marked by an exclusive expression of the key determinant transcription factor, CDX2. Although Hippo signaling pathway is known to be responsible for this TE-specific expression of CDX2, the upstream regulator of this pathway in mammalian embryos is still controversial. In the present study, the involvement of the small molecular G protein, RHOA, in TE cell-fate decision in cattle was investigated. Inhibition of RHOA by the specific inhibitor, C3 transferase (C3), severely impaired the blastocyst formation. Further, C3 treatment significantly decreased the number of blastomeres with nuclearized YAP1, the prominent effector of Hippo pathway. An artificial isolation of ICM cells from blastocysts followed by the continuing culture to regenerate TE cells was conducted and showed that TE re-emergence from the isolated ICM is governed by Hippo pathway and suppressed by C3 treatment like that observed in developing embryos. Finally, the long-term exposure to C3 suggests the presence of alternative regulators of CDX2 expression other than RHOA signaling because there were still CDX2-positive cells after C3 treatment. These results demonstrated that RHOA signaling plays a significant role in TE cell-fate decision by regulating Hippo pathway in cattle.
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http://dx.doi.org/10.1016/j.bbrc.2020.05.210DOI Listing
August 2020

Inverse relationship between autophagy and CTSK is related to bovine embryo quality.

Reproduction 2020 06;159(6):757-766

Research Faculty of Agriculture, Hokkaido University, Hokkaido, Japan.

Improving the quality and the developmental competence of in vitro produced (IVP) embryos is an indispensable goal for assisted reproductive technology. Autophagy is a major protective mechanism for intracellular degradation of unnecessary cytoplasmic components. Autophagy ends by the fusion between autophagic vacuoles and lysosomes, allowing the degradation of the cargo by lysosomal enzymes, especially the cathepsins (CTSs). However, it is still unclear how autophagy and cathepsin K (CTSK) relate to embryo development. This study evaluated (1.) the activities of autophagy and CTSK in relation to bovine embryo quality and (2.) the effect of autophagy induction and/or CTSK inhibition on preimplantation embryo development and quality. We show here that good-quality embryos exhibited a greater autophagic activity and less CTSK activity compared to poor-quality embryos. Blastomeres of an individual embryo may vary in their quality. Good quality blastomeres showed an increased autophagic activity and decreased CTSK activity compared to poor-quality blastomeres within the same embryo at different developmental stages. Importantly, induction of autophagy and/or inhibition of CTSK improved the developmental rate (increased blastocyst and hatching rates) and the quality (increased total cell number and decreased the percentage of apoptotic cells) of IVP bovine embryos. These results demonstrate a promising approach to selectively isolate good-quality embryos and improve the efficiency of IVEP of cattle embryos.
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http://dx.doi.org/10.1530/REP-20-0036DOI Listing
June 2020

Overgrowth of mice generated from postovulatory-aged oocyte spindles.

FASEB Bioadv 2019 Jul 15;1(7):393-403. Epub 2019 Apr 15.

Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture Hokkaido University Sapporo Japan.

Oocyte spindle transfer (OST) is a potent reproductive technology used for mammals that enables the spindle in a deteriorated oocyte at the metaphase of the second meiotic division (MII) to serve as the genetic material for producing descendants. However, whether postnatal growth is achieved via OST using developmentally deteriorated MII oocytes remains unclear. At 16 h after human chorionic gonadotropin administration, denuded MII oocytes immediately after retrieval from oviducts (0 h-oocytes) were used for in vitro fertilization (IVF) as controls. For IVF using postovulatory-aged oocytes, the 0 h-oocytes were further incubated for 12 h and 24 h (12 h- and 24 h-oocytes). These mouse oocytes served as a model for assessing the postnatal growth of individuals produced via OST from developmentally deteriorated oocytes. The embryos from 12 h- and 24 h-oocyte spindles exhibited high rates of development up to the neonatal stage as good as the non-manipulated controls. However, the mice derived from the 24 h-oocyte spindles displayed heavier body weights and greater feed consumption than both controls and mice derived from 12 h-oocyte spindles. Our results demonstrate the feasibility of OST as a potent reproductive technology and its limitation in the use of excessively aged postovulatory oocytes in mammalian reproduction.
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http://dx.doi.org/10.1096/fba.2019-00005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996386PMC
July 2019

Effect of autophagy induction and cathepsin B inhibition on developmental competence of poor quality bovine oocytes.

J Reprod Dev 2020 Feb 25;66(1):83-91. Epub 2019 Dec 25.

Research Faculty of Agriculture, Hokkaido University, Hokkaido 060-8589, Japan.

The present study investigated the effect of autophagy induction and cathepsin B (CTSB) inhibition on developmental competence of poor quality oocytes. Bovine cumulus oocyte complexes (COCs) were classified as good or poor according to their morphology. Autophagy activity was detected in good and poor germinal vesicle (GV) oocytes. Then E-64, a CTSB inhibitor, rapamycin (Rapa), an autophagy inducer, and combined administration was achieved during invitro maturation (IVM) of poor quality COCs followed by detection of autophagy activity. In the next experiment, E-64, Rapa, and E64 + Rapa, were added during IVM to good and poor quality COCs followed by invitro fertilization and culture for 8 days to investigate whether inhibition of CTSB and/or induction of autophagy improve embryonic development and quality. Autophagy activity was significantly lower in poor quality GV oocytes than in good quality ones. E-64, Rapa and E-64 + Rapa treatment during IVM significantly increased autophagy activity in poor quality oocytes. Addition of Rapa in good quality COCs did not increase the blastocyst rate, whereas E-64 increased the blastocyst rate and total cell number (TCN) with decreasing TUNEL-positive cells. In contrast, Rapa treatment in poor quality COCs significantly increased the blastocyst rate and TCN with decreasing TUNEL-positive cells. These results indicate oocyte quality has different responses to intracellular autophagy induction and CTSB activity control by potential autophagy and catabolic status, however, synergetic effect of autophagy induction and CTSB inhibition can increase developmental competence of both good and poor quality COCs, especially rescue effect in poor quality COCs.
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http://dx.doi.org/10.1262/jrd.2019-123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040212PMC
February 2020

Trophectoderm regeneration to support full-term development in the inner cell mass isolated from bovine blastocyst.

J Biol Chem 2019 12 8;294(50):19209-19223. Epub 2019 Nov 8.

Laboratory of Animal Breeding and Reproduction, Research Faculty of Agriculture, Hokkaido University, Kita-ku, Kita 9, Nishi 9, Sapporo 060-8589, Japan

Which comes first: tissue structure or cell differentiation? Although different cell types establish distinct structures delineating the inside and outside of an embryo, they progressively become specified by the blastocyst stage, when two types of cell lineages are formed: the inner cell mass (ICM) and the trophectoderm (TE). This inside-outside aspect can be experimentally converted by the isolation of the ICM from a blastocyst, leading to externalization of the blastomeres composing the outermost layer of the ICM. Here, we investigated the totipotency of isolated mouse and bovine ICMs to determine whether they are competent for TE regeneration. Surprisingly, a calf was generated from the bovine isolated ICM with re-formed blastocoel (re-iICM), but no mouse re-iICMs developed to term. To further explore the cause of difference in developmental competency between the mouse and bovine re-iICMs, we investigated the SOX17 protein expression that is a representative molecular marker of primitive endoderm. The localization pattern of SOX17 was totally different between mouse and bovine embryos. Particularly, the ectopic SOX17 localization in the TE might be associated with lethality of mouse re-iICMs. Meanwhile, transcriptome sequencing revealed that some of the bovine re-iICMs showed transcriptional patterns of TE-specific genes similar to those of whole blastocysts. Our findings suggest that TE regeneration competency is maintained longer in bovine ICMs than in mouse ICMs and provide evidence that the ICM/TE cell fate decision is influenced by structural determinants, including positional information of each blastomere in mammalian embryos.
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http://dx.doi.org/10.1074/jbc.RA119.010746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916479PMC
December 2019

Dynamic status of lysosomal cathepsin in bovine oocytes and preimplantation embryos.

J Reprod Dev 2020 Feb 3;66(1):9-17. Epub 2019 Nov 3.

Graduate School of Global Food Resources(GSF), Hokkaido University, Hokkaido 060-0809, Japan.

Lysosomal cathepsin, in particular cathepsin B (CTSB), plays an important role in implantation, pregnancy, and embryonic development. However, little is known about the mechanism related to the dynamic status of lysosomal cathepsins in bovine oocytes and preimplantation embryos. In the present study, we investigated the dynamics of gene expression, activity, and immunolocalization of CTSB, as well as the activities of lysosome, in bovine oocytes and preimplantation embryos. After gene expression analysis of several cathepsin-related genes, transcript levels of CTSB, CTSD and CTSZ were highest in Metaphase II (MII) oocytes followed by a significant decrease from the 8-cell embryo stage. Activity of CTSB showed a significant increase in 1-cell and morula stage embryos. Lysosomal activity was also significant higher in 1-cell and morula stages, which was consistent with CTSB activities. However, immunolocalization of CTSB did not show the similar pattern of CTSB and lysosomal activities. We also found significantly higher expression levels of CTSB transcript in the trophectoderm (TE) compared to inner cell mass (ICM), whereas activity and immunolocalization of CTSB showed an opposite pattern, i.e. significantly higher in ICM than TE. These patterns were confirmed by the same analysis using separated ICM and TE. Our results suggest that lysosomal CTSB has a pivotal role during embryonic development and differentiation, especially fertilization and the differentiation period.
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http://dx.doi.org/10.1262/jrd.2019-115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040204PMC
February 2020

Effects of use of conventional and sexed semen on the conception rate in heifers: A comparison study.

Theriogenology 2019 Sep 6;135:33-37. Epub 2019 Jun 6.

Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, Kita-ku Kita 9 Nishi 9, Sapporo, 060-8589, Japan. Electronic address:

Conception rate with the use of sexed semen is lower than that with the use of conventional semen, posing a major problem in the dairy industry. The aim of this study was to understand the risk factors that affect the conception rate after artificial insemination (AI) with conventional and sexed semen using field data. The records of the first insemination in Holstein heifers with conventional (n = 41,857) and sexed semen (n = 45,465) in Hokkaido, Japan were analyzed. The mean conception rate after AI from 2012 to 2016 was 56.9% with conventional semen and 47.3% with sexed semen. A multivariable logistic regression model including the effects of year, heifer age, time of the year, semen type, service sire, and their interactions was used to evaluate the interaction effect of heifer age and time of the year by semen type on the conception rate. In the analysis using heifer age, we found that heifers inseminated with sexed semen were approximately 21 days younger than those inseminated with conventional semen. Interestingly, in early, warmer months (Jun, Jul, and Aug), the conception rate after AI with sexed semen significantly decreased compared with that after AI with conventional semen (P < 0.01). Our results showed that more careful implementation of AI is required for a stable conception using sexed semen, particularly during warmer months.
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http://dx.doi.org/10.1016/j.theriogenology.2019.06.012DOI Listing
September 2019

Evaluation of the immune status of peripheral blood monocytes from dairy cows during the periparturition period.

J Reprod Dev 2019 Aug 2;65(4):313-318. Epub 2019 May 2.

Laboratory of Animal Breeding and Reproduction, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.

Calving is a critical but stressful event required for milk production in dairy cows. In the present study, we investigated the immune status of peripheral blood mononuclear cells (PBMCs) isolated from periparturient cows to better understand and, thus, possibly prevent stress during the periparturient period. To evaluate the immune response of PBMCs, we assessed their proliferation with or without a mitogen (concanavalin A, ConA). Blood samples were collected 24 h before and after calving and 1 week after calving. The proliferation of non-treated cells remained unchanged throughout the examination period. The immune response of PBMCs isolated from the cows before calving was relatively low, even after ConA stimulation; however, the immune response of PBMCs collected at both time points after calving was significantly higher than those of non-stimulated controls. Next, we examined the expression patterns of T cell related and inflammatory cytokine genes in PBMCs. We found that the mRNA expression levels of both CD4 and CD8 showed decreasing trends after calving. The expression of the Th1 cell marker gene IFNG also decreased after calving. The mRNA expression level of the inflammatory cytokine gene TNFA increased after parturition. Overall, our results suggest that the PBMC immune response was weakened in cows before delivery and part of the expression of the immune cell-related genes in these cells is altered 24 h before and after calving.
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http://dx.doi.org/10.1262/jrd.2018-150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6708849PMC
August 2019

Significance of CCN2 expression in bovine preimplantation development.

Anim Sci J 2019 Jan 24;90(1):49-54. Epub 2018 Oct 24.

Laboratory of Animal Breeding and Reproduction, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.

In mammalian preimplantation development, the first cell lineage segregation occurs during the blastocyst stage, when the inner cell mass and trophectoderm (TE) differentiate. Species-specific analyses are essential to elucidate the molecular mechanisms that underlie this process, since they differ between various species. We previously showed that the reciprocal regulation of CCN2 and TEAD4 is required for proper TE differentiation in bovine blastocysts; however, the function of CCN2 during early embryogenesis has remained otherwise elusive. The present study assessed the spatiotemporal expression dynamics of CCN2 in bovine embryos, and evaluated how changes to CCN2 expression (using a CCN2 knockdown (KD) blastocyst model) regulate the expression of pluripotency-related genes such as OCT4 and NANOG. The conducted quantitative PCR analysis revealed that CCN2 mRNA was expressed in bovine oocytes (at the metaphase stage of their second meiosis) and embryos. Similarly, immunostaining detected both cytoplasmic and nuclear CCN2 at all analyzed oocyte and embryonic stages. Finally, both OCT4 and NANOG expression levels were shown to be significantly reduced in CCN2 KD blastocysts. Together, these results demonstrate that bovine CCN2 exhibits unique expression patterns during preimplantation development, and is required for the proper expression of key regulatory genes in bovine blastocysts.
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http://dx.doi.org/10.1111/asj.13126DOI Listing
January 2019

Involvement of interferon-tau in the induction of apoptotic, pyroptotic, and autophagic cell death-related signaling pathways in the bovine uterine endometrium during early pregnancy.

J Reprod Dev 2018 Dec 8;64(6):495-502. Epub 2018 Oct 8.

Laboratory of Animal Genetics and Reproduction, Graduate School of Agriculture, Hokkaido University, Hokkaido 060-8589, Japan.

Interferon-tau (IFNT), a type I interferon (IFN), is known as pregnancy recognition signaling molecule secreted from the ruminant conceptus during the preimplantation period. Type I IFNs, such as IFN-alpha and IFN-beta, are known to activate cell-death pathways as well as induce apoptosis. In cows, induction of apoptosis with DNA fragmentation is induced by IFNT in cultured bovine endometrial epithelial cells. However, the status of cell-death pathways in the bovine endometrium during the preimplantation period still remains unclear. In the present study, we investigated the different cell-death pathways, including apoptosis, pyroptosis, and autophagy, in uterine tissue obtained from pregnant cows and in vitro cultured endometrial epithelial cells with IFNT stimulation. The expression of CASP7, 8, and FADD (apoptosis-related genes) was significantly higher in pregnant day 18 uterine tissue in comparison to non-pregnant day 18 tissue. The expression of CASP4, 11, and NLRP3 (pyroptosis-related genes) was significantly higher in the pregnant uterus in comparison to non-pregnant uterus. In contrast, autophagy-related genes were not affected by pregnancy. We also investigated the effect of IFNT on the expression of cell-death pathway-related genes, as well as DNA fragmentation in cultured endometrial epithelial cells. Similar to its effects in pregnant uterine tissue, IFNT affected the increase of apoptosis-related (CASP8) and pyroptosis-related genes (CASP11), but did not affect autophagy-related gene expression. IFNT also increased γH2AX-positive cells, which is a marker of DNA fragmentation. These results suggest that apoptosis- and pyroptosis-related genes are induced by IFNT in the pregnant bovine endometrial epithelial cells.
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http://dx.doi.org/10.1262/jrd.2018-063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305853PMC
December 2018

Hot topic: Pregnancy-induced expression of interferon-stimulated genes in the cervical and vaginal mucosal membranes.

J Dairy Sci 2018 Sep 21;101(9):8396-8400. Epub 2018 Jun 21.

Laboratory of Animal Breeding and Reproduction, Department of Animal Science, Research Faculty of Agriculture, Hokkaido University, Hokkaido 060-8589, Japan. Electronic address:

In ruminants, IFN-tau (IFNT) is a pregnancy recognition signal secreted by the embryonic trophectoderm before implantation, and it induces the expression of IFN-stimulated genes (ISG) in the uterine endometrium and blood leukocytes. The expression of ISG in blood leukocytes could indicate the presence of a viable conceptus before return of the next estrus; however, expression levels have high variation for confirming pregnancy. We hypothesized that the secreted IFNT in the uterus would affect ISG expression in cervical and vaginal tissues because they are directly adjacent to the uterus. To prove the hypothesis, we investigated the expression of 3 ISG (ISG15, MX1, and MX2) in cervical and vaginal mucosal membranes collected from pregnant (n = 12) and nonpregnant (n = 11) lactating Holstein cows at 17 to 18 d after artificial insemination. Mucosal membrane samples of the cervical canal near the external os (cervix) and deep vaginal wall surrounding the external os (vagina) were collected separately by simply scraping with a curette on d 17 or 18 of pregnancy (d 1 = ovulation), at which time IFNT secretion into the maternal uterus is maximal. After pregnancy diagnosis on d 30 and 60, separately collected samples confirmed as pregnant and nonpregnant were used for evaluation of the expression of IFN-stimulated protein 15 kDa (ISG15) and myxovirus-resistance protein 1 and 2 (MX1, MX2) with quantitative real-time PCR. The collected mucosal membrane samples from cervix contained mostly cell clots showing membrane structure and a low content of blood cells. The expression levels of all 3 genes were significantly increased in pregnant cows compared with nonpregnant cows in both cervical and vaginal samples. These results suggest that increased expression of ISG in the cervix and vagina is a pregnancy-associated phenomenon and is highly affected by IFNT secreted from the conceptus through the uterus.
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http://dx.doi.org/10.3168/jds.2017-14251DOI Listing
September 2018

Exchange protein directly activated by cAMP (EPAC) promotes transcriptional activation of the decidual prolactin gene via CCAAT/enhancer-binding protein in human endometrial stromal cells.

Reprod Fertil Dev 2018 Oct;30(11):1454-1461

Department of Endocrine and Neural Pharmacology, Tokyo University of Pharmacy and Life Sciences, Tokyo, 192-0392, Japan.

Protein kinase A (PKA) signalling accompanies elevated intracellular cAMP levels during endometrial stromal cell (ESC) decidualisation. Exchange protein directly activated by cAMP (EPAC), an alternate mediator of cAMP signalling, promotes PKA analogue-induced decidualisation; however, the precise mechanism by which EPAC and PKA co-operatively stimulate decidualisation has not been characterised. To examine the role of CCAAT/enhancer-binding protein (C/EBP) in EPAC- and PKA-mediated decidualisation of primary human ESCs, a reporter plasmid containing the 332bp region upstream from the transcription initiation site of the decidual prolactin (dPRL) gene was generated and the promoter activity was evaluated using a luciferase assay. The dPRL promoter activity was increased by treatment of transfected ESCs with the PKA-selective cAMP analogue N6-phenyl-cAMP (Phe) and enhanced further by co-treatment with the EPAC-selective cAMP analogue 8-(4-chlorophenyltio)-2'-O-methyl cAMP (CPT). Treatment with forskolin, an adenylyl cyclase activator, had a similar effect on reporter activity. Site-directed mutagenesis of the C/EBPβ- and/or C/EBPδ-binding site in the dPRL promoter abolished Phe/CPT-mediated elevation of the reporter activity. EPAC2 knockdown markedly reduced Phe-stimulated C/EBPβ and C/EBPδ mRNA levels, as well as forkhead box O1 (FOXO1) protein levels. These results suggest that EPAC signalling enhances PKA-mediated dPRL expression in ESCs by acting on C/EBP response elements in the promoter region of the gene.
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http://dx.doi.org/10.1071/RD17483DOI Listing
October 2018

Reciprocal regulation of TEAD4 and CCN2 for the trophectoderm development of the bovine blastocyst.

Reproduction 2018 06 16;155(6):563-571. Epub 2018 Apr 16.

Laboratory of Animal Genetics and ReproductionResearch Faculty of Agriculture, Hokkaido University, Sapporo, Japan

The first segregation at the blastocyst stage is the symmetry-breaking event to characterize two cell components; namely, inner cell mass (ICM) and trophectoderm (TE). TEA domain transcription factor 4 (TEAD4) is a well-known regulator to determine TE properties of blastomeres in rodent models. However, the roles of bovine TEAD4 in blastocyst development have been unclear. We here aimed to clarify the mechanisms underlining TE characterization by TEAD4 in bovine blastocysts. We first found that the mRNA expression level was greater in TE than in ICM, which was further supported by TEAD4 immunofluorescent staining. Subsequently, we examined the expression patterns of TE-expressed genes; , and , in the -knockdown (KD) blastocysts. These expression levels significantly decreased in the KD blastocysts compared with controls. Of these downregulated genes, the expression level decreased the most. We further analyzed the expression levels of TE-expressed genes; , and in the KD blastocysts. Strikingly, the KD blastocysts showed the downregulation of , and Furthermore, the ratio of TE-to-ICM cell numbers in the KD blastocysts significantly decreased compared to controls. To our knowledge, this is the first study showing the regulation of expression thorough in mammalian embryos. Not only that, this study also provides evidence that reciprocal regulation of and is required for TE development with appropriate gene expression in bovine blastocysts.
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http://dx.doi.org/10.1530/REP-18-0043DOI Listing
June 2018

Evaluating the electrical impedance and mucus-related gene expression of uterine endometrial tissues in mares.

J Reprod Dev 2018 Apr 7;64(2):193-197. Epub 2018 Jan 7.

Laboratory of Animal Genetics and Reproduction, Department of Animal Science, Research Faculty of Agriculture, Hokkaido University, Hokkaido 060-8589, Japan.

We investigated the electrical impedance of the reproductive tracts (vagina and uterine endometrial tissues) and the expression of mucus-related genes to identify the stage of the estrous cycle in mares. We first examined vaginal impedance in native Hokkaido mares during their estrous cycle and found no significant differences. However, impedance levels tended to decrease towards ovulation. Furthermore, we investigated the estrous cycle by measuring the electrical impedance of the uterine endometrial tissues obtained from carcasses of mares. We found that impedance levels in the endometrial tissues decreased in the regressed phase of the corpus luteum (CL). Expression of mucus-related genes (ATP1A1, CFTR, AQP3, and AQP5) varied at different stages of the estrous cycle. Among them, AQP3 expression was consistent with previous reports. We concluded that electrical impedance in the uterine endometrial tissues of mares could be potentially used to verify the presence of active CL in horses for experimental purposes. However, further studies are needed to determine the reference value and to identify the day of the estrous cycle in mares.
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http://dx.doi.org/10.1262/jrd.2017-128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902908PMC
April 2018

Identification and expression analysis of cDNA encoding insulin-like growth factor 2 in horses.

J Reprod Dev 2018 Feb 17;64(1):57-64. Epub 2017 Nov 17.

Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, Hokkaido 060-8589, Japan.

Insulin-like growth factor 2 (IGF2) is responsible for a broad range of physiological processes during fetal development and adulthood, but genomic analyses of IGF2 containing the 5'- and 3'-untranslated regions (UTRs) in equines have been limited. In this study, we characterized the IGF2 mRNA containing the UTRs, and determined its expression pattern in the fetal tissues of horses. The complete equine IGF2 mRNA sequence harboring another exon approximately 2.8 kb upstream from the canonical transcription start site was identified as a new transcript variant. As this upstream exon did not contain the start codon, the amino acid sequence was identical to the canonical variant. Analysis of the deduced amino acid sequence revealed that the protein possessed two major domains, IlGF and IGF2_C, and analysis of IGF2 sequence polymorphism in fetal tissues of Hokkaido native horse and Thoroughbreds revealed a single nucleotide polymorphism (T to C transition) at position 398 in Thoroughbreds, which caused an amino acid substitution at position 133 in the IGF2 sequence. Furthermore, the expression pattern of the IGF2 mRNA in the fetal tissues of horses was determined for the first time, and was found to be consistent with those of other species. Taken together, these results suggested that the transcriptional and translational products of the IGF2 gene have conserved functions in the fetal development of mammals, including horses.
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http://dx.doi.org/10.1262/jrd.2017-124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830359PMC
February 2018

Novel endogenous retrovirus-derived transcript expressed in the bovine placenta is regulated by WNT signaling.

Biochem J 2017 10 10;474(20):3499-3512. Epub 2017 Oct 10.

Animal Resource Science Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Ibaraki 319-0206, Japan.

Endogenous retroviruses (ERVs) are involved in placentation; perhaps, the most well-known s are the syncytins, actively transcribed genes involved in cell-cell fusion and possible morphological variations. However, ERVs other than syncytins that play an important role in placental development have not been well characterized. To identify genes expressed during the onset of placentation in the bovine species, we characterized the expression profiles of bovine conceptus transcripts during the peri-attachment period using RNA-seq analysis, and confirming some candidates through real-time PCR. Using and PCR analyses, we identified a novel proviral sequence derived from a region, designated bovine endogenous retroviruses (BERV)-K3, containing _p10 and _p24, zinc finger domain. Initial expression of this in bovine conceptuses was on day 20 (day 0 = day of estrus), soon after conceptus attachment to the endometrial epithelium, and its high placental expression was maintained up to the middle of pregnancy. The transcript was also found in the uterine luminal and glandular epithelia, liver, kidney, intestine, and skin. is located on chromosome 7 and integrated within , from which noncoding RNA could be transcribed. Furthermore, the expression of endogenous in bovine trophoblast cell lines was induced by a WNT agonist, a signaling system common to genes expressed in placentas. These data support the argument that during the evolutionary process, mammals incorporated not only similar sequences, but also s unique to individual species. BERV-K3 is in the latter case, likely providing functions unique to ruminant gestation.
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http://dx.doi.org/10.1042/BCJ20170531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633919PMC
October 2017

Enhancement of sperm motility and viability by turmeric by-product dietary supplementation in roosters.

Anim Reprod Sci 2017 Oct 30;185:195-204. Epub 2017 Aug 30.

Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan. Electronic address:

Improving sperm motility and viability are major goals to improve efficiency in the poultry industry. In this study, the effects of supplemental dietary turmeric by-product (TBP) from commercial turmeric production on sperm motility, viability, and antioxidative status were examined in domestic fowl. Mature Rhode Island Red roosters were divided into two groups - controls (groupC) without TBP administration and test subjects (groupT) fed a basal diet supplemented with 0.8g of TBP/day in a temperature-controlled rearing facility (Experiment 1) and 1.6g/day under heat stress (Experiment 2) for 4 weeks. In Experiment 1, TBP dietary supplementation increased the sperm motility variables straight-line velocity, curvilinear velocity, and linearity based on a computer-assisted semen analysis, 2 weeks following TBP supplementation. In Experiment 2, using flow cytometry, sperm viability at 3 and 4 weeks following TBP supplementation was greater in Group T than C, and this increase was consistent with a reduction in reactive oxygen species (ROS) production at 2 and 4 weeks. The results of both experiments clearly demonstrate that dietary supplementation with TBP enhanced sperm motility in the controlled-temperature conditions as well as sperm viability, and reduced ROS generation when heat stress prevailed. Considering its potential application in a range of environments, TBP may serve as an economical and potent antioxidant to improve rooster fertility.
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http://dx.doi.org/10.1016/j.anireprosci.2017.08.021DOI Listing
October 2017

Estrous cycle stage-dependent manner of type I interferon-stimulated genes induction in the bovine endometrium.

J Reprod Dev 2017 Jun 26;63(3):211-220. Epub 2017 Feb 26.

Laboratory of Animal Genetics and Reproduction, Department of Animal Science, Research Faculty of Agriculture, Hokkaido University, Hokkaido 060-8589, Japan.

Interferon tau (IFN-τ) is a ruminant-specific type I IFN secreted by a conceptus before its attachment to the uterus. IFN-τ induces the expression of IFN-stimulated genes (ISGs) via the type I IFN receptor (IFNAR), which is composed of IFNAR1 and IFNAR2 subunits in the endometrium. However, expression patterns of IFNARs during the estrous cycle have not been reported. We hypothesized that the response to a type I IFN changes along with IFNARs and the IFN-regulatory factors (IRFs) driving transcription of IFN signal-related genes and modulating a type I IFN signal during the estrous cycle. We investigated the estrous cycle stage-dependent type I IFN induction of ISGs and expression patterns of IFN signal-related genes in bovine endometrial tissues. Endometrial tissue pieces collected from bovine uteri at each estrous stage (early, mid, and late) were cultured with or without recombinant bovine IFN-α or concentrated pregnant uterine flushing (PUF) on day 18 after confirming the presence of a conceptus. IFN-α and PUF each significantly increased the expression of ISGs in endometrial tissues. The induction levels of the typical ISGs (MX1-a and ISG15) were significantly higher at the mid stage and correlated with high expression of IRFs at the mid stage. The immunostaining of IFNARs showed strong fluorescence intensities in luminal and glandular epithelia at the early and mid stages. Collectively, these results suggest that the endometrium exhibits estrous cycle stage-dependent responsiveness to type I IFN that may be associated with the expression of IFNARs and IRFs for pregnancy recognition.
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http://dx.doi.org/10.1262/jrd.2016-176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481623PMC
June 2017

The Influence of Polyploidy and Genome Composition on Genomic Imprinting in Mice.

J Biol Chem 2016 Sep 16;291(40):20924-20931. Epub 2016 Aug 16.

From the Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589 and

Genomic imprinting is an epigenetic mechanism that switches the expression of imprinted genes involved in normal embryonic growth and development in a parent-of-origin-specific manner. Changes in DNA methylation statuses from polyploidization are a well characterized epigenetic modification in plants. However, how changes in ploidy affect both imprinted gene expression and methylation status in mammals remains unclear. To address this, we used quantitative real time PCR to analyze expression levels of imprinted genes in mouse tetraploid fetuses. We used bisulfite sequencing to assess the methylation statuses of differentially methylated regions (DMRs) that regulate imprinted gene expression in triploid and tetraploid fetuses. The nine imprinted genes H19, Gtl2, Dlk1, Igf2r, Grb10, Zim1, Peg3, Ndn, and Ipw were all unregulated; in particular, the expression of Zim1 was more than 10-fold higher, and the expression of Ipw was repressed in tetraploid fetuses. The methylation statuses of four DMRs H19, intergenic (IG), Igf2r, and Snrpn in tetraploid and triploid fetuses were similar to those in diploid fetuses. We also performed allele-specific RT-PCR sequencing to determine the alleles expressing the three imprinted genes Igf2, Gtl2, and Dlk1 in tetraploid fetuses. These three imprinted genes showed monoallelic expression in a parent-of-origin-specific manner. Expression of non-imprinted genes regulating neural cell development significantly decreased in tetraploid fetuses, which might have been associated with unregulated imprinted gene expression. This study provides the first detailed analysis of genomic imprinting in tetraploid fetuses, suggesting that imprinted gene expression is disrupted, but DNA methylation statuses of DMRs are stable following changes in ploidy in mammals.
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http://dx.doi.org/10.1074/jbc.M116.744144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5076505PMC
September 2016

The Role of Endometrial Selectins and Their Ligands on Bovine Conceptus Attachment to the Uterine Epithelium During Peri-Implantation Period.

Biol Reprod 2015 Aug 1;93(2):46. Epub 2015 Jul 1.

Laboratory of Animal Breeding, Graduate School of Agricultural and Life Science, the University of Tokyo, Tokyo, Japan

A successful pregnancy depends on the blastocyst's implantation to the maternal endometrium; however, the initial interaction between blastocyst and uterine epithelium has not been well characterized. The objectives of this study were to determine if selectins and their ligands were expressed in the bovine conceptus and/or uterus during the periattachment period and to study whether selectins were associated with conceptus attachment to the uterine epithelium. Through the RNA-sequence analysis of bovine conceptuses on Days 17, 20, and 22 (Day 0 = day of estrus), only the SELL ligand, podocalyxin (PODXL), and P-selectin (SELP) ligand, SELPLG, were found. Quantitative PCR analysis confirmed the presence of PODXL and SELPLG in these conceptuses and revealed that SELL, mRNA and protein, detected in the uterine epithelium but not in conceptuses increased during the periattachment period. In the cultured endometrial epithelial cells (EECs), SELL transcript was up-regulated when uterine flushings from Day 20 pregnant animals were placed onto these cells. SELL was also up-regulated when cultured EECs were treated with progesterone, EGF, or bFGF, but not with IFNT. In the coculture system with EECs and bovine trophoblast CT-1 cells, SELL expression in EECs was effectively reduced by its small interfering RNA; however, IFNT, a marker for CT-1 cell attachment to EECs, was not reduced, nor was a transcription factor of IFNT, CDX2. These observations suggest that the conceptus could attach to the uterine epithelium through the use of endometrial SELL and embryonic selectin ligands, possibly initiating the conceptus attachment process in the bovine species.
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http://dx.doi.org/10.1095/biolreprod.115.128652DOI Listing
August 2015
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