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ABSTRACT: We studied the ability of hyaluronan (HA) to inhibit apoptosis in porcine granulosa cells. The granulosa layer with cumulus-oocyte complex is cultured in media supplemented with follicle stimulating hormone (FSH) and 4-MU an inhibitor of hyaluronan synthases. The concentration of HA significantly increased after supplemented with FSH, but significantly decreased with 4-MU. CD44, receptor of HA, expressed after cultured with FSH, decreased in addition low concentration of 4-MU, whereas not detected in high concentration of 4-MU, indicating parallel relation between the amount of HA and CD44 expression. The 4-MU treatment also decreased the expression of procaspase-3, -8, -9 suggesting that inhibition of HA synthesis leads to activation of these caspases. Moreover, addition of anti-CD44 antibody decreased the expression of procaspases suggesting that perturbation of HA-CD44 binding leads activation of caspases. Hence, HA has ability to inhibit apoptosis and HA-CD44 binding is important on apoptosis inhibitory mechanism in porcine granulosa cells.
Biochemical and Biophysical Research Communications 05/2009; 382(1):160-4. · 2.48 Impact Factor
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ABSTRACT: CD44 on macrophages is recognized as a phagocytic receptor involved in the phagocytosis of apoptotic cells. Recently, we detected CD44 on macrophages in atretic follicles during atresia. In this study, we evaluated the distribution of the principal CD44 ligand hyaluronan (HA) and the expressions of HA synthases (HAS: HAS1, HAS2, and HAS3) during atresia in pig ovaries. We determined the 2139-bp sequence of Sus scrofa HAS1 and raised an anti-HAS1 polyclonal antibody. The S. scrofa HAS1 sequence contained six putative HA-binding motifs and conserved amino acid residues crucial for GlcNac transferase activity. HAS1 mRNA expression was upregulated during atresia; however, HAS2 and HAS3 mRNA expression levels were low and very low to undetectable, respectively. Western blotting showed that HAS1 was markedly upregulated during atresia. Immunohistochemical analyses revealed HAS1 distribution in theca cells of healthy and early atretic (stages I and II) follicles and in progressing atretic (stage III) follicles. Hyaluronan was visualized with the HA-binding protein; it accumulated in the theca layer during all stages and in stage III follicles. Hyaluronan assay showed a significantly increased HA concentration in follicular fluid at stage III. Flow cytometry showed HAS1 expression in 55.7% of SIRPA-positive macrophages in stage III follicles. Our results suggest that the HA concentration in follicular fluids increased during atresia and that HAS1 may be the dominant HAS protein in theca cells to produce HA in pig ovaries.
Biology of Reproduction 11/2008; 80(2):249-57. · 4.01 Impact Factor
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ABSTRACT: 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.
Microscopy Research and Technique 07/2006; 69(6):427-35. · 1.79 Impact Factor
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Yuko Miyake,
Hiromichi Matsumoto,
Masaki Yokoo,
Kohtaro Miyazawa,
Naoko Kimura,
Woro Anindito Sri Tunjung,
Takashi Shimizu,
Hiroshi Sasada,
Hisashi Aso,
Takahiro Yamaguchi,
Eimei Sato
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ABSTRACT: 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.
Biology of Reproduction 04/2006; 74(3):501-10. · 4.01 Impact Factor
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ABSTRACT: 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.
Domestic Animal Endocrinology 12/2004; 27(4):397-405. · 2.06 Impact Factor
