Manabu Kawahara

The suppression of fragmentation by stabilization of actin filament in porcine enucleated oocytes

A thorough understanding of the mechanism underlying fragmentation would contribute to the improvement of the developmental ability of reconstructed embryos after nuclear transfer. We conducted the present study to elucidate the influence of the nuclear transfer method on fragmentation of enucleated oocytes and the relationship between change in actin filament distribution and fragmentation. In Experiment 1, we examined activation rates of in vitro matured oocytes. These were 12.9% in maturation alone, 75.7% in electrical stimulation, and 57.9% in ethanol/cycloheximide treatment. In Experiment 2, we observed a higher rate of fragmentation (P < 0.05) in cultured oocytes that had been enucleated and electrically stimulated than in oocytes subjected to the other treatments (maturation alone, enucleation alone and enucleation plus ethanol/cycloheximide activation). In Experiment 3, we stained enucleated and electrically stimulated oocytes with rhodamine/phalloidin dye to show discontinuous distributions in the ooplasm of treated oocytes; oocytes in the other treatment groups showed homogenous distributions of actin filaments (AFs). In Experiment 4, we added cytochalasin B, an inhibitor of AF polymerization, to the culture medium, which prevented fragmentation of enucleated plus electrically stimulated oocytes (cytochalasin B, [+] 0.0%, [-] 60.7% at 24 h after treatment, P < 0.05). In Experiment 5, we investigated the relationship between fragmentation and alteration in AF distribution in enucleated plus electrically stimulated oocytes. At 0 h of culture, enucleated plus electrically stimulated oocytes showed discontinuous distributions of AFs, while nontreated oocytes showed homogenous AF distributions. At 24 and 48 h of culture, fragmentation proceeded in enucleated plus electrically stimulated oocytes and the discontinuous AF distribution diminished with time. In Experiment 6, we added hyaluronic acid (HA) to the culture medium, which suppressed fragmentation of enucleated plus electrically stimulated oocytes (HA, [+] 28.5%, [-] 66.4% at 24 h after treatment, P < 0.05). The results suggest that electrical stimulation induces a change in the AF distribution of oocytes, resulting in fragmentation, and that the addition of HA to the culture media is effective for the suppression of fragmentation.

Expression dynamics of bovine MX genes in the endometrium and placenta during early to mid pregnancy

MX belongs to a family of type I interferon (IFN)-stimulated genes, and the MX protein has antiviral activity. MX has at least two isoforms, known as MX1 and MX2, in mammals. Moreover, bovine MX1 has been found to have alternative splice variants—namely, MX1-a and MX1B. In ruminants, IFN-τ—a type I IFN—is temporarily produced from the conceptus before implantation and induces MX expression in the endometrium. However, the expression dynamics of MX after implantation are not clear. In the present study, we investigated the expression of MX1-a, MX1B and MX2 in the endometrium and placenta before and after implantation along with the expression of IFN-α, type I receptors (IFNAR1 and IFNAR2) and interferon regulatory factors (IRF3 and IRF9). Pregnant uterine samples were divided into five groups according to pregnancy days 14–18, 25–40, 50–70, 80–100, and 130–150. Tissue samples were collected from the intercaruncular endometrium (IC), caruncular endometrium (C) and fetal placenta (P). Although all the MX expressions were significantly higher in the IC and C at days 14–18, presumably caused by embryo-secreted IFN-τ stimulation, their expressions were also detectable in the IC, C and P after implantation. Furthermore, IFN-α expression was significantly higher in the IC. RT-PCR indicated IFNAR1, IFNAR2, IRF3 and IRF9 mRNA in all the tissues during pregnancy. These results suggest that all the MX genes are affected by the type I IFN pathway during pregnancy and are involved in an immune response to protect the mother and fetus.

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

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.

Conserved roles of fibroblast growth factor receptor 2 signaling in the regulation of inner cell mass development in bovine blastocysts

A common process during preimplantation mammalian development is blastocyst formation, which utilizes signaling through fibroblast growth factor receptor 2 (FGFR2), yet the mechanisms through which FGFR2 signaling affect preimplantation development in bovine embryos remain incompletely understood. Here, we used RNA‐interference to investigate the in vitro development, the frequency of blastomere apoptosis, and the mRNA expression of developmental marker genes in FGF receptor 2‐knockdown (FGFR2‐KD) bovine embryos. A reduction in FGFR2 mRNA did not affect preimplantation development or the frequency of apoptotic blastomeres, but did enhanced proliferation of the inner cell mass in blastocysts (P < 0.05)—which differs from the phenotype reported for bovine embryos using a pharmacological approach (treatment with the pan‐FGFR blocker PD173074), but agrees with previous results obtained using mouse embryos. Moreover, the expression of an epiblast marker gene, NANOG, and a primitive endoderm marker gene, GATA6, remained unchanged, whereas the expression of another primitive endoderm marker gene, HNF4A, was significantly reduced in FGFR2‐KD embryos. Therefore, FGFR2 signaling appears to be associated with the regulation of inner cell mass development and proliferation during blastocyst formation in cattle. Mol. Reprod. Dev. 83: 516–525, 2016.

Requirement for nuclear autoantigenic sperm protein mRNA expression in bovine preimplantation development

Nuclear autoantigenic sperm protein (NASP) is associated with DNA replication, cell proliferation, and cell cycle progression through its specific binding to histones. The aim of this study was to examine the roles of NASP in bovine preimplantation embryonic development. Using NASP gene knockdown (KD), we confirmed the reduction of NASP messenger RNA (mRNA) expression during preimplantation development. NASP KD did not affect cleavage but significantly decreased development of embryos into the blastocyst stage. Furthermore, blastocyst hatching was significantly decreased in NASP KD embryos. Cell numbers in the inner cell mass of NASP KD blastocysts were also decreased compared to those of controls. These results suggest that NASP mRNA expression is required for preimplantation development into the blastocyst stage in cattle.

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

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.

Characterization of Toll-Like Receptor 9 expression during Mouse Preimplantation Development

Toll-like receptor 9 (TLR9) is a pathogenic sensing receptor that binds to various pathogenic components, recognizing foreign genomic DNA. Numerous studies have reported the functions of TLR9 in somatic cells, but its feature in germ cells and early embryos has not been well understood. In this study, to characterize TLR9 during preimplantation development, we investigated the mRNA and protein expression dynamics of TLR9 in mouse oocytes and embryos.

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

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.

Significance of CCN2 expression in bovine preimplantation development

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.

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

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 5ʹ- and 3ʹ-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.