Miyake Yuko, Sakurai Masahiro, Tanaka Sachi, Tunjung Woro Anindito Sri, Yokoo Masaki, Matsumoto Hiromichi, Aso Hisashi, Yamaguchi Takahiro, Sato Eimei
Laboratories of Animal Reproduction and Functional Morphology, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan.
Biol Reprod. 2009 Feb;80(2):249-57. doi: 10.1095/biolreprod.108.067694. Epub 2008 Oct 15.
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.
巨噬细胞上的CD44被认为是一种参与凋亡细胞吞噬作用的吞噬受体。最近，我们在闭锁过程中的闭锁卵泡巨噬细胞中检测到了CD44。在本研究中，我们评估了猪卵巢闭锁过程中主要CD44配体透明质酸（HA）的分布以及HA合成酶（HAS：HAS1、HAS2和HAS3）的表达。我们测定了猪HAS1的2139bp序列，并制备了抗HAS1多克隆抗体。猪HAS1序列包含六个假定的HA结合基序和对GlcNac转移酶活性至关重要的保守氨基酸残基。HAS1 mRNA表达在闭锁过程中上调；然而，HAS2和HAS3 mRNA表达水平分别较低和极低至无法检测。蛋白质免疫印迹显示，HAS1在闭锁过程中明显上调。免疫组织化学分析显示，HAS1分布在健康和早期闭锁（I期和II期）卵泡的卵泡膜细胞以及进行性闭锁（III期）卵泡中。用HA结合蛋白观察到透明质酸；它在所有阶段的卵泡膜层以及III期卵泡中积累。透明质酸测定显示III期卵泡液中HA浓度显著增加。流式细胞术显示，III期卵泡中55.7%的SIRPA阳性巨噬细胞表达HAS1。我们的结果表明，卵泡液中的HA浓度在闭锁过程中增加，并且HAS1可能是猪卵巢卵泡膜细胞中产生HA的主要HAS蛋白。
