Biomedical subjects

Masaki Yokoo

Publications and source records attributed to Masaki Yokoo.

13 recordsLinked to original sources

PubMed2006

Role of the hyaluronan receptor CD44 during porcine oocyte maturation.

Previous our studies have shown that CD44, the principal receptor for hyaluronan, is present on cumulus cells during oocyte maturation. Although hyaluronan-CD44 interaction has been implicated in cumulus expansion and/or oocyte maturation, the full significance of CD44 remains unknown. The objective of the present study was to further investigate the role of CD44 in cumulus expansion and oocyte maturation in pigs. We demonstrate here in that CD44 has a key role in oocyte maturation but not in cumulus expansion. Previous studies have reported the physiological significance of cumulus expansion in oocyte maturation. However, our results suggest that cumulus expansion is a necessary condition for oocyte maturation, but that it is not sufficient on its own. Furthermore, western blot analysis demonstrated that the CD44 of the in vitro-matured cumulus-oocyte complexes (COCs) had a larger molecular weight and more terminal sialic acid, which has been proven to inhibit the hyaluronan-binding ability of the receptor, than the CD44 of the in vivo-matured COCs, indicating that the hyaluronan-CD44 interactions during in vitro maturation might be insufficient compared with those in vivo. The insufficient interactions of hyaluronan-CD44 during in vitro maturation may cause the inferior capacity of fertilization and development of oocytes matured in vitro.

Animals↗

PubMed2006

Oxygen consumption of cell suspension in a poly(dimethylsiloxane) (PDMS) microchannel estimated by scanning electrochemical microscopy.

A quantitative analysis of the oxygen concentration profile near a poly(dimethylsiloxane) (PDMS) microfluidic device was performed using scanning electrochemical microscopy (SECM). A microchannel filled with sodium sulfite (Na(2)SO(3)) aqueous solution was imaged by SECM, showing that the oxygen diffusion layer of the PDMS microchannel was observed to be hemicylindrical. Based on a theoretical analysis of the hemicylindrical diffusion layer of the microchannel, the total oxygen mass transfer rates of oxygen to the PDMS microchannel filled with the Na(2)SO(3) solution was calculated to be (4.01 +/- 0.30) x 10(-12) mol s(-1). This is the maximum value of the oxygen transfer rate for this PDMS microchannel device. The oxygen consumption rate increased almost linearly with the logarithm of the concentration of E. coli cells (10(6) approximately 10(8) cells). The respiratory activity for a single E. coli cell was estimated to be approximately 4.31 x 10(-20) mol s(-1) cell(-1).

Cells↗

PubMed2006

Morphodynamics of ovarian follicles during oogenesis in mice.

In the mouse, oogonia enter the prophase of the first meiotic division and differentiate into oocyte while developing in the fetal ovary. Shortly after birth, all oocytes are arrested in the dictyate stage of late prophase in the developing follicles; a small number of follicles reach the ovulatory stage; the rest are lost by apoptosis. The resumption of meiotic division and nuclear progression to metaphase II (oocyte maturation) occur in the ovulatory follicles. In this article we review recent morphological data that have clarified how cytokines and glycosaminoglycans (GAGs) are involved in mouse follicular development, atresia, and maturation during oogenesis, as exogenous/endogenous factors. (1) Microvascular networks and angiogenic factors (epidermal growth factor; GAGs) are deeply involved in selective mouse oocyte growth beyond approximately 20-30 microm in diameter. (2) Gonadotropin-inducible neuronal apoptosis inhibitory protein may indirectly affect oocyte survival as a result of the inhibition of apoptotic granulosa-cell death during folliculogenesis. (3) The pattern of oocyte degeneration depends on follicle and oocyte developmental stages, and follicle stimulating hormone accelerates the process of degeneration of oocytes. (4) The process of degeneration of mouse oocytes/eggs is modulated by tumor necrosis factor-alpha that is accumulated in the expanded cumulus during oocyte maturation. (5) A colloidal iron-positive substance was detected in the intercellular spaces of follicular tissue, especially in the cumulus mass. Cells located where the cumulus mass and granulosa cell layer interwound became enlarged during the resumption of oocyte meiosis. Colloidal iron-positive substances accumulated extensively within the intercellular spaces of the enlarged cells.

Animals↗

PubMed2005

Expression and glycosylation with polylactosamine of CD44 antigen on macrophages during follicular atresia in pig ovaries.

Macrophages are essential in cleaning up apoptotic debris during follicular atresia. However, the key factors of this process are still unclear. In the present study, we evaluated CD44 mRNA, CD44 protein, and CD44 antigen glycosylation on macrophages during follicular atresia in the pig. Atresia was classified into five stages: stage I, healthy follicles; stage II, early atretic follicles having apoptotic granulosa cells with an unclear basement membrane; stage III, progressing atretic follicles having apoptotic granulosa cells completely diffused from the basement membrane; stage IV, late atretic follicles with increasing lysosomal activity; and stage V, disintegrated atretic follicles having collapsed theca cells and strong lysosomal activity. Immunohistological analysis showed that macrophages expressing CD44 invaded the inside of stage III follicles, accompanied by a collapse of basement membrane. Semiquantitative RT-PCR showed that only mRNA of the CD44 standard isoform (CD44s) was present in inner cells of follicles, and not any CD44 variant isoform (CD44v) mRNAs. The amount of CD44s mRNA was increased at stage III. Western blot and lectin blot analyses showed that CD44 was markedly expressed at stage III and glycosylated with polylactosamine at the same time. After macrophages invaded atretic follicles at stages III-V, the CD44 expressed on macrophages was glycosylated with polylactosamine. The lysosomal activity began to increase at stage IV, and reached the highest level at stage V. Increased CD44s protein and posttranslational modification of CD44 with polylactosamine on macrophages from stage III could be involved in the cleaning up apoptotic granulosa cells.

Amino Sugars↗

PubMed2005

Detection of the hyaluronan receptor CD44 in the bovine oviductal epithelium.

Hyaluronan is involved in fundamental reproductive events such as sperm storage in the female reproductive tract, fertilization, and early embryo development, these functions are presumably mediated by its major cell surface receptor, CD44. The present study was conducted to investigate the presence and localization of CD44 in the bovine oviductal epithelium, using immunohistochemical and Western blot methods on tissue sections and epithelial cell extracts collected from the uterotubal junction (UTJ), isthmus, and ampulla of animals in the oestrus or luteal phase of the oestrous cycle. While positive immunolabelling for CD44 was found on the ad-luminal surface and supra-nuclear region of epithelial cells in all tubal segments investigated, in the UTJ, there were epithelial cells in which the entire cytoplasm positively stained. We found no differences in terms of CD44-positive staining between the different stages of the oestrous cycle. Presence of CD44 was detected by Western blotting in the tubal epithelium as a single band at 200 kDa. Although it appeared in all tubal segments, the expression of CD44 protein was more accentuated in the sperm reservoir (UTJ) than in the other segments. This is the first time CD44 has been detected in the epithelium of the tubal sperm reservoir in cattle, suggesting a pathway for the action of hyaluronan in this segment.

Animals↗

PubMed2005

Hyaluronan and its binding proteins in the epithelium and intraluminal fluid of the bovine oviduct.

Hyaluronan (HA) is involved in several important steps of sperm storage and of fertilization. This study investigates the presence and concentration of HA in oviductal fluid (ODF), together with the localization of HA and the presence of hyaluronan-binding proteins (HABPs) in the oviductal epithelium of normally cycling dairy heifers and cows. The concentration and amount of HA in ODF, collected over the course of several oestrous cycles via catheters placed in the isthmic and ampullar tubal segments, were measured using an ELISA. The concentration and amount of HA in ODF did not vary significantly between these anatomical regions, nor between the stages of the oestrous cycle (p > 0.05), although the amount of HA seemed to peak during oestrous. The most HA per day (2.9 +/- 0.64 microg, least square mean +/- SEM) was produced on the day of ovulation, whereas the lowest amount (1.25 +/- 0.68 microg) was produced 4 days before ovulation. To investigate the localization of HA, tissue samples were retrieved at well-defined stages of the oestrous cycle and from corresponding regions of the oviduct. Sections and protein extracts from the tissue samples were studied histochemically using biotinylated HABP and immunoblotted with fluorescein isothiocyanate (FITC)-HA, respectively. Presence of HA labelling in the oviductal epithelium was restricted to the sperm reservoir, a localization that seemed to be cycle-independent. The immunoblotting of samples from the lining epithelium revealed seven bands of HABPs. We confirm that the bovine oviduct produces HA and its binding proteins, and that HA is mainly localized to the epithelium of the sperm reservoir.

Animals↗

PubMed2004

Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes.

Phosphatidylinositol 3-kinase (PI3K) is known to play critical roles in signal transduction processes related to a variety of cellular activities. In the present study, we investigated the role of PI3K during meiotic maturation in mouse oocytes using a specific inhibitor, LY294002. In follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest of cumulus oocyte complexes (COCs), LY294002 suppressed germinal vesicle breakdown (GVBD), first polar body (PB1) emission, and cumulus expansion. To examine the effect of LY294002, denuded oocytes (DOs) were cultured in medium containing follicular fluid meiosis-activating sterol (FF-MAS) since absence of gonadotropin receptors in oocytes has been reported and FSH did not stimulate meiotic maturation of DOs in the presence of hypoxanthine. In FF-MAS-induced maturation of DOs, LY294002 suppressed PB1emission, but not GVBD. In spontaneous gonadotropin-independent oocyte maturation, LY294002 had no effect on COCs and DOs. Akt/protein kinase B, a serine-threonine kinase, is a key downstream effector of the PI3K pathway. Therefore, we also examined the distribution of Akt during FSH-induced meiotic maturation. The distribution of Ser(473) phosphorylated Akt was similar to the localization of microtubules, while Thr(308) phosphorylated Akt was present in the pericentriolar materials (PCM) in metaphase I (MI) and II (MII) oocytes. LY294002 decreased the amount of Thr(308) phosphorylated Akt to very low to undetectable levels in MI and MII oocytes. Ser(473) phosphorylated Akt showed aberrant distribution and very low to undetectable levels of expression in LY294002-treated MI and MII oocytes, respectively. These results suggest that PI3K and Akt participate in mouse meiotic maturation.

Animals↗

PubMed2004

Differential expression of bone morphogenetic protein 4-6 (BMP-4, -5, and -6) and growth differentiation factor-9 (GDF-9) during ovarian development in neonatal pigs.

Growth differentiation factor-9 (GDF-9) and bone morphogenetic proteins (BMPs), comprise the largest subgroups of ligands in the TGF-beta superfamily, and have been shown to be involved in follicle development in mammals. However, whether these factors are involved in folliculogenesis in pigs is still unknown. The present study was performed to determine the relationships between early folliculogenesis and the expression of GDF-9 and BMP (BMP-4, -5 and -6) mRNAs in neonatal pigs. Ovaries were removed at 5, 16, 28 and 39 days after birth to examine the follicular population (the right ovary of each animal) and to detect mRNA expression (the left ovary of each animal). Primordial follicles accounted for >80% of the ovarian follicles from 5 days until 39 days after birth. A marked increase in primary follicles and the appearance of secondary follicles were observed in the ovaries at 28 days after birth. BMP-4, -5, and -6 and GDF-9 mRNAs were expressed by ovaries at 5-, 16-, 28- and 39-day-old pigs. The peak expression of BMP-4, -5, and -6 and GDF-9 mRNAs was observed in the ovaries at 5, 39, 28 and 16 days, respectively, after birth. These data demonstrate that folliculogenesis in piglets might be controlled by the interaction with these factors. We conclude that BMPs and GDF-9 may have distinct functions in several stages of follicle development in neonatal pig ovaries.

Animals↗

PubMed2004

Cumulus-oocyte complex interactions during oocyte maturation.

In most mammals, the oocyte in the Graafian follicle is surrounded by tightly packed layers of cumulus cells, forming the cumulus-oocyte complex. During the preovulatory period, cumulus cells change from a compact cell mass into a dispersed structure of cells for the synthesis and deposition of a mucoid intercellular matrix, a process referred to as cumulus expansion. Cumulus expansion is thought to influence a variety of fundamental developmental changes during oocyte maturation. Volumetric expansion of the cumulus-oocyte complex correlates, at least in pig, with the outcome of oocyte maturation, fertilization, and embryo development. Therefore, detailed functional studies of cumulus expansion seem to be required to elucidate the mechanism of oocyte maturation. We summarize the current knowledge about (1) morphological changes of cumulus-oocyte complexes during oocyte maturation, (2) follicle factors inducing cumulus expansion, (3) the role of cumulus expansion in oocyte maturation, (4) cytoplasmic regulators of oocyte maturation, and (5) possible roles of cumulus expansion.

Animals↗

PubMed2004

Molecular cloning of porcine growth differentiation factor 9 (GDF-9) cDNA and its role in early folliculogenesis: direct ovarian injection of GDF-9 gene fragments promotes early folliculogenesis.

Growth differentiation factor-9 (GDF-9) is a growth factor secreted by oocytes in growing ovarian follicles. To investigate the ovarian function of GDF-9 in pigs, we first cloned porcine GDF-9 complementary DNA (cDNA), and then injected its gene fragments into the ovary in gilts. Porcine GDF-9 has open reading frame (ORF) homologies of 81.4%, 84.6%, 84.2%, 72.7% and 72.6% with its human, bovine, ovine, rat and mouse counterparts respectively. Regarding the deduced amino-acid sequence of the mature protein, the corresponding homologies reach 92.1%, 97.8%, 97.0%, 89.6% and 88.1% respectively. To investigate the role of GDF-9 in early folliculogenesis, the ovaries of 2-month-old prepubertal gilts were injected with GDF-9 gene fragments. The injection of porcine GDF-9 gene fragments resulted in an increase in the number of primary, secondary and tertiary follicles, concomitant with a decrease in the number of primordial follicles. These results indicated that exogenous GDF-9 can promote early folliculogenesis in the porcine ovary, and that a technique for direct ovarian injection of GFD-9 gene fragments may contribute to a novel therapy for prevention and treatment of infertility associated with ovarian dysfunction.

Amino Acid Sequence↗

PubMed2003

Follicular microvasculature and angiogenic factors in the ovaries of domestic animals.

The genetic and molecular mechanisms that control the development of capillary blood vessels during follicular development are beginning to be elucidated. Ovarian follicles contain and produce angiogenic factors that may act alone or in concert to regulate the process of thecal angiogenesis. These factors are ultimately controlled by endocrine, paracrine and autocrine regulation. A recent study indicated that vascular endothelial growth factor (VEGF) plays an important role in the process of thecal angiogenesis during follicular development. We are developing a novel technology for the induction of follicular development using the technique of in vivo gene administration. Here, we summarize the recent progress of our research.

Animals↗

PubMed2002

Localisation of the hyaluronan receptor CD44 in porcine cumulus cells during in vivo and in vitro maturation.

Polyspermy is fairly common during porcine in vitro fertilisation (IVF), perhaps due to incomplete in vitro oocyte maturation (IVM). Porcine cumulus cells (CCs) layered around the oocyte produce large amounts of extracellular hyaluronan (HA) when forming an expanding cell cloud during the last phase of oocyte maturation. The specific actions of HA are mediated via HA-binding proteins (HABPs), such as CD44, which act as receptors. In this study using immunocytochemistry and western blotting we investigated the localisation of CD44 in CCs obtained from in vivo-matured pig cumulus-oocyte complexes (COCs) and compared it with that in CCs from immature COCs and of COCs subjected to IVM and IVF procedures. Immunolabelling of CD44 was absent or very weak in CCs from immature COCs but strongly present on the surface of the CCs obtained from in vivo, displaying a similar localisation in the in vitro-matured COCs. In the latter, the labelling decreased but did not disappear in CCs 4 h after sperm co-incubation during IVF. Immunoblotting detected bands of between 73 and 88 kDa, corresponding to CD44, in the protein extract from in vivo CCs collected immediately prior to, or following spontaneous ovulation. The in vitro-matured CCs, however, presented bands ranging from 81 kDa to 88 kDa. Also, the bands found in the in vivo-matured CCs showed a larger variation of intensity and migration among animals than did the batches of in vitro-matured CCs. No CD44 band was detected on aliquots of the frozen-thawed boar spermatozoa used for IVF. The results clearly demonstrate that the specific HA receptor CD44 is present in expanding CCs of in vivo-matured pig COCs, in relation to increasing amounts of inter-CC HA. The subtle differences in molecular weight and migration ability observed between in vivo and in vitro samples may relate to differences in glycosylation and thus explain differences in HA-binding ability, of consequence for optimising in vitro culture conditions.

Animals↗

PubMed2002

Identification of hyaluronic acid-binding proteins and their expressions in porcine cumulus-oocyte complexes during in vitro maturation.

Hyaluronic acid-binding proteins (HABPs) are necessary for expansion of the cumulus-oocyte complex (COC) during oocyte maturation. In this study, to obtain the detailed information of HABPs during cumulus expansion, we examined the expression of HABPs in porcine COCs during in vitro maturation (IVM). After maturation culture, proteins were extracted from porcine COCs and separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes. After transfer, the membranes were subjected to ligand blotting with biotinylated hyaluronic acid (bHA) or fluorescein isothiocyanate-labeled hyaluronic acid (FITC-HA). Furthermore, the extracted proteins were subjected to immunoprecipitation, Western blotting, and immunofluorescence analysis to dissect the HABPs. Ligand blotting with FITC-HA could detect HABPs. Using this ligand-blotting method, 13 and 14 bands of HABPs were detected in porcine COCs after 0 and 48 h in culture, respectively. Of these, the level of expression of 85-kDa HABP increased with cumulus expansion during IVM and was newly detected after culture. Immunoprecipitation, Western blotting, and immunofluorescent analysis confirmed that the 85-kDa HABP corresponded to CD44 and that it existed on/in the membrane of cumulus cells. The present results indicated that HABP expressed in porcine COCs during IVM, particularly CD44, may form a network of the matrices in the extracellular space of the oocyte with cumulus expansion during IVM.

Animals↗