Atsushi Ideta

Involvement of GATA transcription factors in the regulation of endogenous bovine interferon‐Tau gene transcription

Expression of interferon‐tau (IFNT), necessary for pregnancy establishment in ruminant ungulates, is regulated in a temporal and spatial manner. However, molecular mechanisms by which IFNT gene transcription is regulated in this manner have not been firmly established. In this study, DNA microarray/RT‐PCR analysis between bovine trophoblast CT‐1 and Mardin–Darby bovine kidney (MDBK) cells was initially performed, finding that transcription factors GATA2, GATA3, and GATA6 mRNAs were specific to CT‐1 cells. These mRNAs were also found in Days 17, 20, and 22 (Day 0 = day of estrus) bovine conceptuses. In examining other bovine cell lines, ovary cumulus granulosa (oCG) and ear fibroblast (EF) cells, GATA2 and GATA3, but not GATA6, were found specific to the bovine trophoblast cells. In transient transfection analyses using the upstream region (−631 to +59 bp) of bovine IFNT gene (bIFNT, IFN‐tau‐c1), over‐expression of GATA2/GATA3 did not affect the transcription of bIFNT‐reporter construct in human choriocarcinoma JEG3 cells. Transfection of GATA2, GATA3, ETS2, and/or CDX2, however, was effective in the up‐regulation of the bIFNT construct transfected into bovine oCG and EF cells. One Point mutation studies revealed that among six potential GATA binding sites located on the upstream region of the bIFNT gene, the one next to ETS2 site exhibited reduced luciferase activity. In CT‐1 cells, endogenous bIFNT gene transcription was up‐regulated by over‐expression of GATA2 or GATA3, but down‐regulated by siRNA specific to GATA2 mRNA. These data suggest that GATA2/3 is involved in trophoblast‐specific regulation of bIFNT gene transcription. Mol. Reprod. Dev. 76: 1143–1152, 2009.

Expression of mesenchymal-related genes by the bovine trophectoderm following conceptus attachment to the endometrial epithelium

In the course of experiments to identify and characterize the factors that function in bovine conceptuses during peri-attachment periods, various transcripts related to the epithelial-mesenchymal transition (EMT) were found. In this study, RNA was extracted from different sets of days 17, 20, and 22 (day 0=day of estrous) bovine conceptuses and subjected to real-time PCR analysis as well as Western blotting, from which abundances of N-cadherin (CDH2), vimentin, matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) (MMP2), and matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) (MMP9) mRNAs were determined on day 22, concurrent with (CDH1) mRNA and protein downregulation. Transcription factors in EMT processes were then analyzed and changes in snail homolog 2 (Drosophila) (SNAI), zinc finger E-box binding homeobox 1 (ZEB1), zinc finger E-box binding homeobox 2 (ZEB2), twist homolog 1 (Drosophila) (TWIST1), twist homolog 2 (Drosophila) (TWIST2), and Kruppel-like factor 8 (KLF8) transcripts were found in day 22 conceptuses, while confirming SNAI2 expression by Western blotting. Immunohistochemical analysis revealed that the day 22 trophectoderm expressed the mesenchymal markers N-cadherin and vimentin as well as the epithelial marker cytokeratin. In attempts to identify the molecular mechanisms by which the trophectoderm expressed EMT-related genes, growth factor receptors associated with EMT were analyzed. Upregulation of the growth factor receptor transcripts, fibroblast growth factor receptor 1 (FGFR1), platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), platelet-derived growth factor receptor, beta polypeptide (PDGFRB), and transforming growth factor, beta receptor II (70/80 kDa) (TGFBR2) mRNAs, was found on day 22. The analysis was extended to determine the integrin (ITG) transcripts and found high levels of integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) (ITGA4), integrin, alpha 8 (ITGA8), integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) (ITGB3), and integrin, beta 5 (ITGB5) mRNAs on day 22. These observations indicate that after the conceptus-endometrium attachment, EMT-related transcripts as well as the epithelial marker cytokeratin were present in the bovine trophectoderm and suggest that the implantation process for noninvasive trophoblasts requires not only extracellular matrix expression but also partial EMT.

Function of a Transcription Factor CDX2 Beyond Its Trophectoderm Lineage Specification

The transcription factor caudal-related homeobox 2 (CDX2) regulates trophectoderm differentiation, but its function beyond trophectoderm differentiation is not well characterized. CDX2 was shown to regulate a trophoblast-specific gene, interferon τ (IFNT), in the ruminants. However, its regulatory mechanism has not been determined. Here, we report a new role of CDX2 in histone modifications of the IFNT gene. Chromatin immunoprecipitation assays using ovine conceptuses obtained from d 14, 16, 16.5, or 20 of pregnancy (d 0, day of mating) revealed that H3K18 acetylation was highly detectable at the upstream and open reading frame regions of the IFNT gene on d 14 and 16, when CDX2 reached its peak expression. From d 16.5, when the conceptus initiates attachment to uterine epithelial cells, histone acetylation along with CDX2 expression declines. Two candidate CDX2 binding sites (-300 to -294 bp and -293 to -287 bp) of the bovine IFNT gene promoter region were detected from chromatin immunoprecipitation and luciferase assay. When Cdx2 constructs were transfected into bovine ear-derived fibroblast cells, histone acetylation was increased, concurrent with the recruitment of cAMP response element binding protein-binding protein, which has histone acetyltransferase activity. H3K18 acetylation was seen in the proximity of the CDX2 binding region located at the IFNT genes upstream region in CT-1 cells, but when these cells were treated with specific CDX2 small interfering RNA, H3K18 acetylation was decreased. These findings suggest that CDX2 regulates its targeted gene through cAMP response element binding protein-binding protein recruitment, which correlates with greater histone acetylation.

Administration of peripheral blood mononuclear cells into the uterine horn to improve pregnancy rate following bovine embryo transfer.

Embryo transfer (ET) has been used to improve reproductive efficiency and genetic make-up in bovine species. However, the success rate of ET has not been improved since its inception. Here we examined whether administration of autologous peripheral blood mononuclear cells (PBMCs) into the uterine horn can improve pregnancy rates following bovine ET. First we determined that the abundance of interleukin (IL)-1alpha, IL-1beta and IL-8 transcripts in PBMCs was greatest after 24h of culture. PBMCs that had been cultured for 24h were gently administered non-surgically to the uterine horn ipsilateral to the corpus luteum on day 4 of the estrous cycle. On day 7, the ET was carried out and the pregnancy rate in the PBMC-treated group was compared with that in the non-treated group. The pregnancy rate on day 60 in the PBMC-treated group (76.7%, 56/73) was significantly higher than that in the non-treated group (59.7%, 43/72, p<0.05). These results indicate that administration of autologous PBMCs into the uterine horn improves pregnancy rates following bovine ET.

Involvement of VCAM1 in the bovine conceptus adhesion to the uterine endometrium

Following bidirectional communication, the conceptus and the uterine epithelium must establish a proper cell-cell interaction, resulting in the progression of implantation processes. To clarify the mechanism of conceptus attachment to the uterine endometrium, we studied whether vascular cell adhesion molecule (VCAM1) was expressed in bovine conceptuses or endometrium during the peri-attachment period. Uterine VCAM1 expression was minimal in day 17 (day 0=day of estrus) cyclic and pregnant animals, but increased between days 20 and 22 of pregnancy. In the intercaruncular regions, VCAM1 protein was localized to the luminal and glandular epithelia, whereas in the caruncular regions, VCAM1 protein was detected in the stroma and endothelia of the uterine endometrium. In cultured endometrial epithelial cells (EECs), VCAM1 expression was up-regulated when treated with uterine flushings or growth factor and further increased when EECs were cocultured with bovine trophoblast CT1 cells. VCAM1 expression in CT1 cells was also up-regulated with the use of uterine flushings, and further increased when these cells were cocultured with EECs. Expression of VCAM1 receptor, integrin α 4 (ITGA4) mRNA, increased significantly in day 22 conceptuses. In day 22 pregnant uteri, VCAM1 protein was found in both EECs and conceptuses, but ITGA4 was localized only to trophoblasts. These observations indicate that cell-cell interactions between conceptuses and uterine epithelial cells are required for sufficient VCAM1 and ITGA4 expression in the bovine species and suggest that uterine VCAM1 and conceptus ITGA4 play a role in the establishment of conceptus adhesion to the uterine endometrium.

Intrauterine administration of peripheral blood mononuclear cells enhances early development of the pre‐implantation bovine embryo

Intrauterine administration of peripheral blood mononuclear cells (PBMCs) prior to bovine embryo transfer (ET) was previously shown to improve the pregnancy rate. To better understand how PBMCs improve the pregnancy rate, we examined gene expression in the cells from uterine lumen and evaluated the morphology of bovine pre‐attachment embryos in utero following intrauterine administration of PBMCs. On day 3 of the estrous cycle (day 0 = estrous), bovine PBMCs were isolated and suspended in RPMI 1640, and were incubated for 24 hr. The cultured PBMCs were administered non‐surgically to the uterine horn ipsilateral to the corpus luteum on day 4 of the estrous cycle (PBMC group). On day 9, endometrial–luminal lymphoid cells from uterine lumen ipsilateral to the corpus luteum were collected by uterine flushing. Transcripts for macrophage‐colony stimulating factor in the lymphoid cells were more abundant in the PBMC group than in the control group (P < 0.05). On day 7 (of the separate experiments), five blastocysts were each transferred to the luminal area, to which PBMCs had been administered on day 4. These embryos were allowed to develop in utero until day 15 of gestation, when embryos were non‐surgically retrieved from the uterus. The average length of trophoblasts recovered from the PBMC group was significantly longer than that of the control group (51.6 ± 7.8 vs. 27.4 ± 6.0 mm, P < 0.05). Our results strongly suggest that intrauterine administration of PBMCs improves endometrial environment, which promotes early development of pre‐attachment conceptuses. Mol. Reprod. Dev. 77:954–962, 2010.

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

ABSTRACT A successful pregnancy depends on the blastocysts 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.

Ionotropic glutamate receptor AMPA 1 is associated with ovulation rate

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1Asn has a weaker affinity to glutamate than GRIA1Ser, both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1Asn have a slower luteinizing hormone (LH) surge than cows with GRIA1Ser. In addition, cows with GRIA1Asn possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1Ser. Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy.

Down‐regulation of interferon tau gene transcription with a transcription factor, EOMES

Interferon tau (IFNT), produced for a short interval during early pregnancy by the ruminant embryonic trophectoderm, is essential for the maintenance of early pregnancy, but the mechanism by which it is transcriptionally regulated has not been fully determined. To identify a transcription factor(s) involved in the down‐regulation of IFNT genes, mRNAs for various known transcription factors were investigated by reverse‐transcriptase and real‐time PCR in conceptus tissues collected on Days 15, 17, and 21, or Days 17, 20, and 22 of ovine or bovine pregnancy, respectively. In particular, the T‐box protein eomesodermin (EOMES) exhibited high mRNA expression in Day 17 or 22 ovine or bovine conceptuses. Interaction between EOMES and the identified transcription factors was studied using transient transfection, revealing that ovine/bovine IFNT‐reporter transactivation was down‐regulated by EOMES. Transcription factor interactions with EOMES were further studied through immunoprecipitation, demonstrating an association between EOMES and cAMP‐response element binding protein (CREB)‐binding protein (CREBBP). Uterine flushing media collected from cyclic or early pregnancy animals were added to bovine trophoblast CT‐1 cells cultured on type‐I collagen (monoculture) or bovine uterine epithelial cells (coculture) in an attempt to regulate EOMES expression. In the coculture, but not the monoculture, addition of uterine flushing from Day 17 pregnant animals resulted in increased EOMES expression in CT‐1 cells. These results suggest that as conceptuses attach to the uterine epithelium, IFNT gene transcription is down‐regulated by an increase in EOMES expression and EOMES–CREBBP binding in the attached trophoblast cells. Mol. Reprod. Dev. 80: 371–383, 2013.

A simple medium enables bovine embryos to be held for seven days at 4°C

Cryopreservation methods using liquid nitrogen (LN2) for gametes and embryos are prevalent in mammalian artificial reproduction. However, the pregnancy rate from frozen embryos has not improved over the past two decades because freeze–thawing causes significant damage. The strict regulation of transportation of LN2 containers by airlines also limits exchange between breeders. In this article, we introduce a medium that enabled bovine embryos to be held for up to 7 days at 4°C. A pregnancy rate of 75% (24/32) was obtained for embryos held for 7 days in this medium and transferred to primed recipients. Its constituents were medium 199, foetal bovine serum, and HEPES for buffering. This technique will enable LN2-free storage and air transportation of embryos provided transplantation to recipients can be completed within 7 days.