Misa Hosoe

Conditions that affect acquisition of developmental competence by mouse oocytes in vitro: FSH, insulin, glucose and ascorbic acid.

The simplest unit required for the support of oocyte growth and development is the oocyte-granulosa cell complex. Therefore, a culture system was established that utilizes these complexes to assess mechanisms promoting nuclear, cytoplasmic and genomic maturation in mammalian oocytes. Deletion of serum from the culture, results in increased apoptosis in oocyte-associated granulosa cells (OAGCs), however, addition of ascorbic acid (0.5 mM) significantly reduced the level of apoptosis in the OAGCs, although no improvement of oocyte developmental competence was detected. The effects of reducing glucose during oocyte growth were studied since, under some culture conditions, glucose has deleterious effects on early preimplantation development. Reducing the glucose concentration to 1 mM resulted in the production of oocytes with greatly reduced developmental competence. Deleterious effects of FSH plus insulin during oocyte growth in vitro on preimplantation development are reviewed and discussed in terms of the communication of oocytes with inappropriately developing granulosa cells. Evidence that oocytes promote the appropriate differentiation of OAGCs in intact follicles in vivo is also discussed. It is hypothesized that oocytes control the differentiation of these cells, in order to promote intercellular signaling essential for the acquisition of competence to undergo normal embryogenesis.

Global gene expression analysis and regulation of the principal genes expressed in bovine placenta in relation to the transcription factor AP-2 family

BackgroundCell-cell communication is an important factor in feto-maternal units during placentogenesis. The placenta produces pivotal hormones and cytokines for communication between cotyledonary villi and the maternal caruncle. Gene expression in bovine placenta throughout pregnancy was comprehensively screened by a cDNA microarray, and we searched for a common transcription factor in a gene cluster that showed increasing expression throughout gestation in cotyledonary villi and caruncle.MethodsPlacentomal tissues (villi and caruncle) were collected from Day 25 to Day 250 of gestation for microarray analysis. Global gene expression profiles were analyzed using the k-means clustering method. A consensus sequence cis-element that may control up-regulated genes in a characteristic cluster was examined in silico. The quantitative expression and localization of a specific transcription factor were investigated in each tissue using quantitative real-time RT-PCR and in situ hybridization.ResultsThe microarray expression profiles were classified into ten clusters. The genes with most markedly increased expression became concentrated in cluster 2 as gestation proceeded. Cluster 2 included placental lactogen (CSH1), pregnancy-associated glycoprotein-1 (PAG1), and sulfotransferase family 1E estrogen-preferring member 1 (SULT1E1), which were mainly detected in giant trophoblast binucleate cells (BNC). Consensus sequence analysis identified transcription factor AP-2 binding sites in some genes in this cluster. Quantitative real-time RT-PCR analysis confirmed that high level expression of transcription factor AP-2 alpha (TFAP2A) was common to cluster 2 genes during gestation. In contrast, the expression level of another AP-2 family gene, transcription factor AP-2 beta (TFAP2B), was extremely low over the same period. Another gene of the family, transcription factor AP-2 gamma (TFAP2C), was expressed at medium level compared with TFAP2A and TFAP2B. In situ hybridization showed that TFAP2A, TFAP2B and TFAP2C mRNAs were localized in trophoblast cells but were expressed by different cells. TFAP2A was expressed in cotyledonary epithelial cells including BNC, TFAP2B was specifically expressed in BNC, and TFAP2C in mononucleate cells.ConclusionWe detected gestational-stage-specific gene expression profiles in bovine placentomes using a combination of microarray and in silico analysis. In silico analysis indicated that the AP-2 family may be a consensus regulator for the gene cluster that characteristically appears in bovine placenta as gestation progresses. In particular, TFAP2A and TFAP2B may be involved in regulating binucleate cell-specific genes such as CSH1, some PAG or SULT1E1. These results suggest that the AP-2 family is a specific transcription factor for clusters of crucial placental genes. This is the first evidence that TFAP2A may regulate the differentiation and specific functions of BNC in bovine placenta.

Development of the competence of bovine oocytes to release cortical granules and block polyspermy after meiotic maturation

Bovine immature oocytes do not have the ability to block polyspermic penetration. The present study was conducted to determine whether this is correlated to cortical granule (CG) distribution and the competence of oocytes to release CG upon sperm penetration, and whether the ability of bovine oocytes to release CG develops during in vitro maturation. Fluorescein isothiocyanate‐conjugated Lens culinaris agglutinin was used for detecting CG in immature and mature oocytes before and after sperm penetration and electric stimulation. The labeled oocytes were examined with laser confocal and fluorescent microscopes. The results show that CG exist as clusters in all immature oocytes. The CG were not released from immature oocytes exposed to electric pulse or penetrated by spermatozoa, resulting in 94% of oocytes being polyspermic. When immature oocytes were cultured for 22h in vitro, 81% extruded the first polar body and reached metaphase II. In mature oocytes, 25% of oocytes showed CG clusters, 42% and 33% of oocytes showed partial and complete CG dispersion, respectively. When mature oocytes were inseminated in vitro, only 15% of oocytes were polyspermic. Cortical granule exocytosis occurred in 97% of oocytes after sperm penetration and 84% of oocytes released all of the CG 18 h after insemination. Electric pulse induced all of the mature oocytes to release CG but only 55% released all of their CG 18 h post stimulation. These results indicate that polyspermy in immature bovine oocytes is the result of the complete failure of the oocyte to release CG after sperm penetration. Bovine oocytes became competent to release CG by sperm penetration and electric stimulation after meiotic maturation. These results provide evidence that CG exocytosis plays an important role(s) in the establishment of the block to polyspermy in bovine oocytes.

Differential neutrophil gene expression in early bovine pregnancy

BackgroundIn food production animals, especially cattle, the diagnosis of gestation is important because the timing of gestation directly affects the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection or the accuracy, simplicity, or cost of the method. A new method for detecting gestation, which involves assessing interferon-tau (IFNT)-stimulated gene expression in peripheral blood leukocytes (PBL), was recently proposed. PBL fractionation methods were used to examine whether the expression profiles of various PBL populations could be used as reliable diagnostic markers of bovine gestation.MethodsPBL were collected on days 0 (just before artificial insemination), 7, 14, 17, 21, and 28 of gestation. The gene expression levels of the PBL were assessed with microarray analysis and/or quantitative real-time reverse transcription (q) PCR. PBL fractions were collected by flow cytometry or density gradient cell separation using Histopaque 1083 or Ficoll-Conray solutions. The expression levels of four IFNT-stimulated genes, interferon-stimulated protein 15 kDa (ISG15), myxovirus-resistance (MX) 1 and 2, and 2′-5′-oligoadenylate synthetase (OAS1), were then analyzed in each fraction through day 28 of gestation using qPCR.ResultsMicroarray analysis detected 72 and 28 genes in whole PBL that were significantly higher on days 14 and 21 of gestation, respectively, than on day 0. The upregulated genes included IFNT-stimulated genes. The expression levels of these genes increased with the progression of gestation until day 21. In flow cytometry experiments, on day 14 the expression levels of all of the genes were significantly higher in the granulocyte fraction than in the other fractions. Their expression gradually decreased through day 28 of gestation. Strong correlations were observed between the expression levels of the four genes in the granulocyte fractions obtained with flow cytometry and with density gradient separation.ConclusionsThe expression profiles of ISG15, MX1, MX2, and OAS1 could be a useful diagnostic biomarker of bovine gestation. Assessing the expression levels of these genes in a granulocyte fraction obtained with density gradient separation is a practical way of detecting gestation in cows within three weeks of insemination.

Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles

BackgroundBovine follicular development is regulated by numerous molecular mechanisms and biological pathways. In this study, we tried to identify differentially expressed genes between largest (F1) and second-largest follicles (F2), and classify them by global gene expression profiling using a combination of microarray and quantitative real-time PCR (QPCR) analysis. The follicular status of F1 and F2 were further evaluated in terms of healthy and atretic conditions by investigating mRNA localization of identified genes.MethodsGlobal gene expression profiles of F1 (10.7 +/- 0.7 mm) and F2 (7.8 +/- 0.2 mm) were analyzed by hierarchical cluster analysis and expression profiles of 16 representative genes were confirmed by QPCR analysis. In addition, localization of six identified transcripts was investigated in healthy and atretic follicles using in situ hybridization. The healthy or atretic condition of examined follicles was classified by progesterone and estradiol concentrations in follicular fluid.ResultsHierarchical cluster analysis of microarray data classified the follicles into two clusters. Cluster A was composed of only F2 and was characterized by high expression of 31 genes including IGFBP5, whereas cluster B contained only F1 and predominantly expressed 45 genes including CYP19 and FSHR. QPCR analysis confirmed AMH, CYP19, FSHR, GPX3, PlGF, PLA2G1B, SCD and TRB2 were greater in F1 than F2, while CCL2, GADD45A, IGFBP5, PLAUR, SELP, SPP1, TIMP1 and TSP2 were greater in F2 than in F1. In situ hybridization showed that AMH and CYP19 were detected in granulosa cells (GC) of healthy as well as atretic follicles. PlGF was localized in GC and in the theca layer (TL) of healthy follicles. IGFBP5 was detected in both GC and TL of atretic follicles. GADD45A and TSP2 were localized in both GC and TL of atretic follicles, whereas healthy follicles expressed them only in GC.ConclusionWe demonstrated that global gene expression profiling of F1 and F2 clearly reflected a difference in their follicular status. Expression of stage-specific genes in follicles may be closely associated with their growth or atresia. Several genes identified in this study will provide intriguing candidates for the determination of follicular growth.

Quantitative analysis of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) gene expression in calf and adult bovine ovaries

BackgroundIt has been reported that calf oocytes are less developmentally competent than oocytes obtained from adult cows. Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) play critical roles in folliculogenesis, follicular development and ovulation in mammalian ovaries. In the present study, we attempted to compare the expression patterns of BMP15 and GDF9 in the cells of calf and cow ovaries to determine a relationship between the level of these genes and the low developmental competence of calf oocytes.MethodsBovine tissues were collected from 9-11 months-old calves and from 4-6 years-old cows. We characterized the gene expression of BMP15 and GDF9 in calf and adult bovine oocytes and cumulus cells using quantitative real-time reverse transcriptase polymerase chain reaction (QPCR) and in situ hybridization. Immunohistochemical analysis was also performed.ResultsThe expression of BMP15 and GDF9 in cumulus cells of adult ovaries was significantly higher than that in calf ovaries, as revealed by QPCR. GDF9 expression in the oocytes of calf ovaries was significantly higher than in those of the adult ovaries. In contrast, BMP15 expression in the oocytes of calf and adult ovaries was not significantly different. The localization of gene expression and protein were ascertained by histochemistry.ConclusionsOur result showed for the first time BMP15 and GDF9 expression in bovine cumulus cells. BMP15 and GDF9 mRNA expression in oocytes and cumulus cells was different in calves and cows.

Activation of protein kinase C induces cortical granule exocytosis in a Ca2+-independent manner, but not the resumption of cell cycle in porcine eggs

The effects of protein kinase C (PKC) activation on meiotic resumption and cortical granule (CG) exocytosis as well as its dependence on Ca2+ in porcine eggs matured in vitro were studied. Cortical granule release was judged by both confocal laser microscopy after the eggs were labeled with fluorescein isothiocyanate‐peanut agglutinin (FITC‐PNA) and electron microscopy. Meiotic resumption and pronuclear formation were observed after eggs were stained with acetic orcein. When eggs were treated with PKC activators, 1‐oleyl‐2‐acetyl‐glycerol (OAG) or phorbol 12‐myristate 13‐acetate (PMA), the pronuclear formation percentage was significantly lower than that of Ca2+ ionophore A23187‐treated group, but not statistically different from that in negative control group (P > 0.05), and most of the eggs were still arrested at metaphase II stage, suggesting that PKC activation does not induce the resumption of meiosis and pronuclear formation. In contrast, PKC activation induced 89.1% to 100% of the eggs completely or partially released their CG in different groups, not statistically different from A23187‐treated group, and this effect could be overcome by PKC inhibition. When the intracellular free Ca2+ was chelated with acetoxymethal ester form of 1,2‐bis(0‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA‐AM), and then treated with PMA or OAG in Ca2+‐free medium, the proportions of eggs with CG release were 90.9% and 78.1%, respectively, not statistically different from the above‐treated groups, suggesting that CG exocytosis induced by PKC activation is independent of Ca2+ rise. The results indicate that different events of porcine egg activation may be uncoupled from one another.

Cloning of the Bovine Antiapoptotic Regulator, BCL2-Related Protein A1, and Its Expression in Trophoblastic Binucleate Cells of Bovine Placenta

Abstract This report studied the identification and sequence of a full-length cDNA for the bovine BCL2 antiapoptotic family member, BCL2-related protein A1 (BCL2A1), and its localized and quantitative expression in the placenta to clarify the regulatory mechanism of trophoblast cell proliferation and differentiation during implantation and placental development. We cloned a full-length bovine BCL2A1 cDNA with 725 nucleotides and an open-reading frame corresponding to a protein of 175 amino acids. The predicted amino acid sequence shared 78% homology with human BCL2A1. All BCL2 homology domains (BH1, BH2, BH3, and BH4) in bovine BCL2A1 were conserved as well as in other mammalian BCL2A1. In the placentomes, in situ hybridization demonstrated that the BCL2A1 was limited in binucleate cells expressing various pregnancy-specific molecules like placental lactogen. BCL2-associated X protein (BAX) was also expressed in binucleate cells. Quantitative real-time RT-PCR detection exhibited a high-level expression of BCL2A1 in the conceptus at Day 21 of gestation, and it was expressed and increased in the extraembryonic membrane, cotyledon, and intercotyledon from implantation to term. BAX expression intensity increased with progression of gestation and remained elevated in postpartum. Caspase-3 protein (CASP3) and mRNA (CASP3) were detected from late gestation to postpartum in placenta as well as in the results of TUNEL detection. We believe that the apoptosis of binucleate cells may be regulated by the balance of the BCL2A1 and BAX. BCL2A1 genes produced a BCL2A1 protein in the mammalian cell-expression system. This molecule is a new candidate for antiapoptotic maintenance of the binucleate cells that support placental functions throughout gestation in bovine.

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle

BackgroundExtracellular matrix metalloproteinase inducer (EMMPRIN) regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow.MethodsIn this study, endometrial tissues from the cyclic cows during before ovulation, after ovulation and middle of estrous cycle; and pregnant endometrial tissues from Day 19 to 35 of gestation have been used. Expression of mRNA was analyzed by RT-PCR, qPCR and in situ hybridization whereas protein expression by immunohistochemistry and western blot analysis.ResultsEMMPRIN mRNA was expressed in both cyclic and pregnant endometrium and significantly higher in the endometrium at Day 35 of gestation than the cyclic endometrium. In Western blot analysis, an approximately 65 kDa band was detected in the endometrium, and approximately 51 kDa in the cultured bovine epithelial cells and BT-1 cells, respectively. Both in situ hybridization and immunohistochemistry data showed that EMMPRIN was primarily expressed in luminal and glandular epithelium with strong staining on Day 19 conceptus. At Day 19 of gestation, expression of EMMPRIN mRNA on luminal epithelium was decreased than that observed at middle of estrous cycle, however, on Day 30 of gestation, slightly increased expression was found at the site of placentation. Expression of matrix metalloproteinase-2 (MMP-2) and MMP-14 mRNA were mainly detected in stroma and their expression also decreased at Day 19 of gestation however it was also expressed at the site of placentation at Day 30 of gestation as observed for EMMPRIN. Expression of MMP-1 or -9 mRNA was very low and was below the detection limit in the cyclic and pregnant endometrium.ConclusionEMMPRIN from the luminal epithelium may regulate the expression of stromal MMP-2 and -14 suggesting its crucial role in adhesion and fusion of embryo to luminal epithelium by directly itself through physiological tissues remodeling and developmental process, and/or stimulating MMPs to compensate endometrial functions.

Characterization and expression analysis of SOLD1, a novel member of the retrotransposon-derived Ly-6 superfamily, in bovine placental villi.

Background Ly-6 superfamily members have a conserved Ly-6 domain that is defined by a distinct disulfide bonding pattern between eight or ten cysteine residues. These members are divided into membrane-type and secretory-type proteins. In the present study, we report the identification of a novel Ly-6 domain protein, secreted protein of Ly-6 domain 1 (SOLD1), from bovine placenta. Principal Findings SOLD1 mRNA was expressed in trophoblast mononucleate cells and the protein was secreted into and localized in the extracellular matrix of the mesenchyme in cotyledonary villi. SOLD1 bound mainly with type I collagen telopeptide. We confirmed secretion of SOLD1 from the basolateral surface of a bovine trophoblast cell line (BT-1). It may be related to the organization of the extra-cellular matrix in the mesenchyme of fetal villi. Since trophoblast mononucleate cells are epithelial cells, their polar organization is expected to have a crucial role in the SOLD1 secretion system. We established that SOLD1 is an intronless bovine gene containing the Alu retrotransposon, which was integrated via cytoplasmic reverse transcription. Conclusion We identified a novel retrotransposon-like Ly-6 domain protein in bovine placenta. SOLD1 is a crucial secreted protein that is involved in the organization of the mesenchyme of the cotyledonary villi. Furthermore, the gene encoding SOLD1 has an interesting genomic structure.