{"paper_id":"300e67ae-38e5-4f72-9e9d-4c9af6cdb87b","body_text":"Expression and Distribution of Cyclooxygenase-2\nin Human Ovary during Follicular Development\n言語: English\n出版者: OSAKA CITY MEDICAL CENTER\n公開日: 2018-07-04\nキーワード: COX-2, Ovulation, Follicular development,\nIVF-ET\n作成者: Tokuyama, Osamu, 中村, 嘉宏, Musoh, Ayako,\n本田, 謙一, 尾崎, 宏治, 石河, 修\nメールアドレス: \n所属: Osaka City University, Osaka City University,\nOsaka City University, Osaka City University, Osaka City\nUniversity, Osaka City University\nメタデータ\nhttps://ocu-omu.repo.nii.ac.jp/records/2020144URL\n\nTokuyama Osamu, Nakamura Yoshihiro, Musoh Ayako, Honda Ken-ichi, Ozaki Koji, Ishiko Osamu. \nExpression and Distribution of Cyclooxygenase-2 in Human Ovary during Follicular Development. \nOsaka City Medical Journal. 2003, 49, 1, 39-47. \nExpression and Distribution of \nCyclooxygenase-2 in Human Ovary during \nFollicular Development \n  \nTokuyama Osamu, Nakamura Yoshihiro, Musoh Ayako, Honda \nKen-ichi, Ozaki Koji, Ishiko Osamu \n \nCitation Osaka City Medical Journal. 49(1); 39-47 \nIssue Date 2003-06 \nType Journal Article \nTextversion Publisher \nRight \n© Osaka City Medical Association. \nhttps://osakashi-igakukai.com/.  \n \n \nPlaced on: Osaka City University Repository \n\nReceived  November  15,  2002;  accepted  November  26,  2002.\nCorrespondence to: Osamu Tokuyama, MD. \nDepartment of Obstetrics and Gynecology, Osaka City University Medical School, \n1-4-3, Asahi-machi, Abeno-ku, Osaka, 545-8585, Japan\nTel: +81-6-6645-3861;   Fax: +81-6646-5800\nE-mail: otoku@med.osaka-cu.ac.jp\n-39 -\nOsaka City Med.  J. Vol. 49, \u0014\u001a-\u0015\u0018, 2003\nExpression and Distribution of Cyclooxygenase-2 in Human Ovary\nduring Follicular Development\nOSAMU TOKUYAMA,Y OSHIHIRO NAKAMURA,A YAKO MUSOH,K EN-ICHI HONDA,\nKOJI OZAKI, and OSAMU ISHIKO\nDepartment of Obstetrics and Gynecology, Osaka City University Medical School\nSummary\nProstaglandins play important roles for oocyte maturation and follicular rupture.\nCyclooxygenase-2 (COX-2), an inducible isoform of a prostaglandin metabolic enzyme, is\nessential for the timed production of prostaglandins in the ovary.  The aim of the present study\nis to examine expression and distribution of COX-2 in human ovarian follicles at each of the\nmaturation stages.  Immunohistochemical staining shows that COX-2 is expressed in the\ngranulosa cell layer of secondary and developing follicles, but is not detected in primary and\nGraafian follicles.  Western blotting analysis revealed the existence of COX-2 in periovulatory\nfollicular fluid at a mean concentration of 5.6 \nʶ 0.6 ng/ml.  COX-2 might begin to be produced at\nthe secondary follicle stage, and once a follicle reaches the Graafian follicle stage, the production\nof this enzyme is stopped.  After exposure to luteinizing hormone (LH) surge, follicles might\nresume production of COX-2, and this is secreted into the follicular fluid. \n,FZ\u00018PSET\u001bCOX-2; Ovulation; Follicular development; IVF-ET\nIntroduction\nFollicular development and ovulation is a critical process in mammalian female reproduction,\nand depends largely upon the harmonized effects of pituitary gonadotropins, follicular\nstimulating hormone (FSH), and luteinizing hormone (LH)\n1).  However, how gonadotropins\nregulate this complex reproductive process is not fully clarified, though some gonadotropin\neffects are mediated by other mediators.  Among these mediators, prostaglandins, the\ncyclooxygenase (COX) metabolites of arachidonate, play a crucial role in this process.  \nDuring the reproductive period, primordial follicles are irregularly scattered throughout a\nnarrow band in the superficial ovarian cortex.  A primordial ovarian follicle consists of a\nprimordial oocyte and a thin monolayer follicle epithelium.  As the oocyte increases cellular\nvolume the follicular epithelium becomes cuboidal; this stage is called the primary follicle stage.\nProgression from the primordial stage to the primary follicle stage is not in an FSH dependent\n\nmanner, though the exact mechanism is still unclear.  However, once a follicle reaches this\nstage, it develops in a FSH dependent manner.  Under the influence of FSH, the granulosa cell\nlayer becomes thickened, multilayered.  This stage is called the secondary follicle stage, and\nduring it a theca cell layer is formed surrounding the granulosa cell layer.  The diameter of a\nfollicle reaches about 0.2 to 0.4 mm, and storage of follicular fluid begins to be detected in an\nantral space.  This stage is called the antral follicle stage.  After formation of the antral space,\nthe follicle continues to grow to a diameter of 4.0 mm to 6.0 mm.  These size follicles have been\nrecruited in the mid-luteal phase of the previous menstrual cycle and the growth of the recruited\nfollicles is stimulated by the elevated FSH levels in the late luteal to early follicular phases.\nUsually one follicle that succeeds in gaining a superior estrogen secretion ability is selected from\namong the cohort and is called the dominant follicle.  LH surge, which is induced by the\nelevation of the serum estrogen concentration, evokes follicular rupture.  The ruptured follicle\ntransforms into corpus lutea that produce progesterone to induce secretory change in the uterine\nendometrium and to sustain implantation.  All these processes are largely controlled by\ngonadotropins, but how these hormones regulate these processes remains unelucidated.  It has\nbeen reported that some gonadotropin actions are mediated by mediators other than hormonal\nones.  Prostaglandins (PG), putative intraovarian mediators playing a crucial role in this\nprocess, are produced and metabolized by the action of the enzyme, cyclooxygenase\n2).\nCyclooxygenase, which converts arachidonic acid into PGs, has two types of isoforms,\ncyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)\n3-5).  COX-1 is the constitutive form and\nCOX-2 the inducible form 6-8).  The latter is thought to be essential for oocyte maturation and\nfollicular rupture.  In rodents or primates, expression and distribution of COX-2 in ovarian\nfollicles has been reported, but there are no reports on human.  Our study aims to examine\nexpression and distribution of COX-2 in human ovarian follicles at each maturation stage, and\nin follicular fluid.\nMaterials and methods\nOvarian tissue\nOvarian tissues were obtained from women receiving gynecological surgery at Osaka City\nUniversity Hospital, after informed consent was given by all the patients included in this study.\nTissues were fixed in 10% buffered formalin and embedded in paraffin.\nImmunohistochemistry\nSections (5 µm) from the paraffin-embedded tissues were deparaffinized with xylene, hydrated\nwith ethanol and water, and then immersed in 3% hydrogen peroxide for 5 min to block\nendogenous peroxidase activity.  Sections were washed in Phosphate Buffer Saline (PBS) and\nincubated in PBS containing 10% normal goat serum blocking solution for 20 min to block non-\nspecific binding.  These were then reacted with anti-human COX-2 rabbit IgG (Immuno-\nBiological Laboratories Gunma, Japan) 1:200 diluted with blocking solution for 24 h at 4\n, and\nwashed 3 times with 0.02% Tween 20 in PBS.  The sections were next reacted with anti-rabbit\nIgG 1:200 diluted with blocking solution for 1 hour and washed 3 times with 0.02% Tween 20 in\nPBS.  Immunocomplexes were colored with 0.2 mg/ml diaminobenzidine (DAB), 0.1% hydrogen\nperoxide in 0.05 M Tris-HCl buffer.  Immunostaining was ascertained by two observers using\nTokuyama et al\n-40 -\n\nlight microscopy.\nIn vitro fertilization and embryo transfer\nThe subjects were 40 patients who underwent in-vitro fertilization and embryo transfer (IVF-\nET) due to tubal factors.  Patients with endocrinological abnormalities, which included polycystic\novaries and hyperprolactinemia, or endometriosis or complications of autoimmune diseases,\nwere excluded.  Patients were given gonadotropin releasing hormone agonist (Gn-RHa) to suppress\npituitary function and received controlled ovarian hyperstimulation.  The administration of\ntransnasal nafarelin acetate (Nasanyl, Yamanouchi Japan Tokyo, Japan) was initiated at mid-\nluteal phase.  After down regulation of gonadotropins was sufficient, ovarian stimulation using\nhuman menopausal gonadotropin (hMG) (humegon: Organon Japan, Tokyo, Japan) was initiated on\nthe fifth day of menstruation.  Follicle diameter was measured by transvaginal ultrasound, and\nwhen the dominant follicle reached at least 16-18 mm in maximum diameter, 10,000 units of\nhuman chorionic gonadotropin (hCG) (Mochida Japan, Tokyo, Japan) was administered\nintramuscularly.  After 36 hours from the administration of hCG, follicular puncture and\naspiration of follicular fluid was performed under transvaginal ultrasound using a 16-gauge-\nneedle (COOK, Brisbane, Australia).  Pure follicular fluid obtained from the dominant follicle\nwas centrifuged immediately at 1,800 rpm for 10 minutes at room temperature.  The\nsupernatants were stored at -80\nuntil assayed.  \nImmunoaffinity column chromatography with anti human COX-2 rabbit IgG\nFollicular fluid (0.3 ml)stored at -80\n was diluted with 0.7 ml of 10 mM Tris-HCl (pH 7.5),\npassed through a blue HiTrap column (0.7 ʷ 2.5 cm, Amersham Pharmacia, Buckinghamshire,\nEngland) for exclusion of albumin.  The eluate was reacted with HiTrap NHS-activated HP\nSepharose (Amersham Pharmacia, Buckinghamshire, England) coupled with anti human COX-2\nrabbit IgG, and the adsorbed protein was eluted with 0.1 M glycine-HCl buffer (pH 2.5) and\ndialyzed against 10 mM Tris-HCl buffer (pH 7.6) overnight.  The dialyzed protein was freeze-\ndried until required for Western blotting analysis.\nWestern blotting analysis of COX-2 in follicular fluid\nThe freeze-dried protein was dissolved with 0.05 ml sodium dodecyl sulfate (SDS) sample\nbuffer, heated at 95\n for 3 min and separated by 10% SDS polyacrylamide sodium dodecyl\nsulfate (SDS) gel.  The separated proteins in the gel were transferred to polyvinylidene\ndifluoride (PVDF) membrane (Hybond-P, Amersham Pharmacia, Buckinghamshire, England).\nThe blotted membrane was reacted with 1:500 diluted anti-human COX-2 rabbit IgG (Immuno-\nBiological Laboratories, Gunma, Japan), washed with PBS containing 0.1% Tween-20, and\nreacted with anti-rabbit immunoglobulin goat IgG 1:5,000 diluted with PBS containing 20% fetal\nbovine serum.  The immuno-complexes were detected with an ECL system on Hyperfilm ECL\n(Amersham Pharmacia).\nCOX-2 concentrations in follicular fluid\nCOX-2 concentrations in follicular fluid were measured using a Human COX-2 Enzyme\nImmuno Assay KIT (Immuno-Biological Laboratories).  Intra-and inter-assay coefficients of\nvariation (CV) were less than 10%.  Data were analyzed for age, number of retrieved oocytes, the\ntotal dose of hMG preparation and follicular estradiol, and for progesterone concentration.\nCOX-2 Expression during Follicular Development \n-41 -\n\nE2 and P4 concentrations in follicular fluid\nEstradiol (E2) and progesterone (P4) concentrations in follicular fluid were measured\nutilizing radioimmunoassay (Diagnostic Products Corporation, Los Angeles, USA).  Intra- and\ninterassay coefficients of variation were less than 10%.  \nStatistical analysis\nData are expressed as mean ʶ SD unless otherwise indicated.  The correlation were analyzed\nby simple regression analysis using software package statview.\nResults\nImmunohistochemistry in ovarian tissue\nCOX-2 immunostaining was examined in follicles at four stages of follicular maturation.  In\nprimary follicles, COX-2 could not be detected in the granulosa cell layer.  However, at the\nsecondary follicle stage, COX-2 protein could be detected in the granulosa cell layer.  The\nexpression of the COX-2 was uniform throughout the granulosa cell layer.  In developing or\nTokuyama et al\n-42 -\nFigure 1.  Immunohistochemistry in ovarian tissue (A: original magnification ʷ 400, B and C:\noriginal magnification ʷ 200, D: original magnification ʷ 100).  No COX-2 expression was\nfound in the primary follicle (A) and the Graafian follicle (D).  COX-2 expression was\nfound in the secondary follicle (B) and developing follicle (C) (black arrows).\nFigure 2.  Immunohistochemistry in developing follicle.  COX-2 expression in the developing follicle\nwas found with anti-COX-2 IgG (B) but not with IgG blocked with antigen (A) (A and B:\noriginal magnification\nʷ 400).\n\nantral follicles, COX-2 expression was also recognized, and again the distribution of this enzyme\nwas uniform throughout granulosa cell layer.  Sections of Graafian follicles before exposure to\nLH surge did not show immunoreactivity to COX-2 antibodies (Fig. 1 and 2).  A lack of exposure\nto LH surge was verified by semiquantitative detection of urinary LH. \nClinical background of patients undergoing IVF-ET\nTable-1 shows the clinical background of patients who underwent IVF-ET.  Figures in this\ntable were expressed as mean ʶ standard deviation (SD).  Mean patient age was 33.5 ʶ 0.6\nyears.  Mean total hMG dose administered was 2058.1 ʶ 132.6 IU/ml and the mean number of\noocytes retrieved was 7.8 ʶ 1.1. \nWestern blotting and COX-2 concentrations in follicular fluid\nDue to the high concentration of the albumin, exclusion of the albumin through Sepharose\ncolumn and dialysis is an indispensable step to immunodetection of COX-2 in follicular fluid.\nThe COX-2 protein in follicular fluid sample from an IVF-ET patient showed about 70,000\nmolecular mass units (Fig. 3).  The mean COX-2 concentration in follicular fluid was 5.6\nʶ 0.6\nng/ml (range: 0.6-19.5 ng/ml).  Significant correlations were not found for COX-2 and age,\noocytes retrieved, total hMG. \nEndocrinological background of patients\nThe mean E2 and P4 concentrations in follicular fluid from 16 patients were 179250.0 ʶ\nCOX-2 Expression during Follicular Development \n-43 -\nmean ʶ\u0001SD  \nAge (years) 33.5  ʶ\u00010.6 (range: 26.0 to 42.0)\nTotal dose of hMG (IU/ml) 2058.1  ʶ\u0001132.6 (range: 600.0 to 5400.0)\nOocytes retrieved   7.8  ʶ\u00011.1 (range: 1.0 to 32.0)\nTable 1.  40 patients background: age, total hMG, and oocytes retrieved\nFigure 3.  Western blot analysis of protein from follicular fluid shows existence of COX-2.  The\nmolecular masses of standard proteins separated on the same gel are indicated on the\nright side (lane 3).  The band showing existence of COX-2 corresponds to a molecular\nweight of about 70,000 (lane 1 and 2).\n\n118953.0 pg/ml and 6639.0 ʶ 3194.0 ng/ml, respectively (Table 2).  Correlation was found\nbetween COX-2 and E2 concentration in follicular fluid (R=0.538, p<0.05), but not between COX-\n2 and P4 (Fig. 4).\nDiscussion\nIn this study, we demonstrated that COX-2 protein begins to be expressed at the secondary\nfollicle stage in human ovary and that this expression is withdrawn just before exposure to LH\nsurge.  We also measured COX-2 concentrations in follicular fluid after administration of hCG,\nwhich can substitute for the LH surge function in patients who have received Gn-RHa\ndesensitization against the pituitary gland.  We also verified the presence of COX-2 in follicular\nfluid after exposure to hCG in IVF-ET patients.\nOocyte maturation and follicular rupture largely depend on coordinated secretion of pituitary\ngonadotropins and many mediators are involved in follicular maturation and rupture, with\nprostanoids considered to play a crucial role.  Previous studies demonstrated that COX-2, but\nnot COX-1, is a major source of PGs in primate and other mammalian species\n9).  Among these\nCOX-2 metabolites, PGE2 and F2 α are especially considered to play pivotal roles in follicular\ndevelopment and rupture10).\nEarly experiments on rodents showed that PGE2 was involved in activation of the cumulus\noocyte complex 11), which is characterized by polarization and expansion of the granulosa cells\nsurrounding the oocyte with production of hyaluronic acid-enriched proteoglycans 12).  This\nprocess is considered to be critical for successful fertilization and follicular rupture.  Oocytes\nobtained from COX-2 deficient mice showed a severely compromised fertilization rate\n13), and\nEP2, a PGE2 receptorsubtype, deficient mice also showed a much reduced fertilization rate 14).\nDavis et al reported that PGE2 supplementation can restore cumulus activation in COX-2\ndeficient mice\n15).  In bovine, Calder et al reported that COX-2 and PGE2 play very essential roles\nin the cumulus oocyte complex 16).  These lines of reporting suggest that COX-2 derived PGE2 is\nTokuyama et al\n-44 -\nFigure 4.  Correlation between COX-2 and E2 concentrations in follicular fluid.  There was correlation\nbetween COX-2 and E2 (R=0.538, p<0.05) (A), but not between COX-2 and P4 (B). \nmean ʶ\u0001SD    \nCOX-2 (ng/ml) 3.6  ʶ\u00011.5 (range: 0.6 to 6.5)\nE2 (pg/ml) 179250.0  ʶ\u0001118953.0 (range: 44500.0 to 493000.0)\nP4 (ng/ml) 6639.0  ʶ\u00013194.0 (range: 2640.0 to 14000.0)\nTable 2.  COX-2, E2 and P4 concentrations in follicular fluid of 16 patients\n\nrequired for oocyte cumulus complex maturation in ovarian follicles.  In our study, COX-2 began\nto be detected at the secondary follicle stage and continued to be detected in granulosa cell\nlayers of antral follicles.  This finding suggests that COX-2 and the metabolites of this enzyme\nmight be involved in follicular development including oocyte cumulus complex maturation in\nhuman. \nCOX-2 and its metabolites are also essential in follicular rupture, which is induced by LH\nsurge and has a process similar to an inflammatory reaction Recent reports demonstrated that\nprostaglandins, especially PGE2 and PGF2\nα , play critical roles in the events of follicular rupture\nand that COX-2 is a essential enzyme to produce these metabolites.  Administration of non-\nselective COX inhibitors to monkeys has been shown to have an adverse effect on follicular\nrupture, and this inhibitory effect is reversed by the administration of PGF2\nα 17).  Recently, the\nsame effect of COX inhibitors was reported in human 18).  COX-2 knockout mice have a defect in\nfollicular rupture, which can be restored by the exogenous administration of PGE2 15).  Sirois\ndemonstrated that COX-2 expression by granulosa cells and prostaglandin concentrations in follicular\nfluid of preovulatory follicles rose dramatically in response to the ovulatory gonadotrophin surge\n19).\nThe interval between the onset of the ovulatory gonadotrophin surge and follicular rupture varies in\nmammalian species.  Sirois and Dore proposed that elevated COX-2 activity and the resulting increase\nin follicular fluid PG determine the timing of ovulation in mammals\n20).  In our study, preovulatory\nfollicles before the LH surge did not show any signals in immunohistochemical study, and the same\nobservation was reported in preovulatory rhesus monkey follicles before LH surge\n21).  We speculated\nthat temporal withdrawal of COX-2 in the granulosa cells layer of Graafian follicles before exposure \nto LH surge is partially explained by the follicles’ readiness to respond to LH surge and the\nsubsequent ovulatory events in which COX-2 and its metabolites play pivotal roles.  However,\nfurther intensive study is required to elucidate this temporal withdrawal of the enzyme.\nWe previously reported follicular COX-2 concentrations in 20 women\n22); and this time we\nexpand the subject base to 40 patients.  All patients included in this study received pituitary\nsuppression by Gn-RHa, and superovulatory treatment using hMG.  LH surge which induces\nCOX-2 in follicles and induces resumption of the meiosis oocyte is substituted by hCG.  However,\nthe COX-2 concentrations in this study might not reflect COX-2 concentrations in natural\novulatory follicles, as previous studies demonstrated COX-2 induction in the granulosa cell layer\nin preovulatory follicles after the exposure to LH surge.  COX-2 in follicular fluid is considered to\nbe secreted from granulosa cells after HCG stimulation.  COX-2 concentrations and E2 showed a\npositive relationship in our study, and this finding suggested that the COX-2 concentration in\nfollicular fluid could be a candidate as a novel indicator for the function of granulosa cells.  We\ncould not determine any relationship between COX-2 concentration and P4 levels in follicular\nfluid. \nWe obtained follicular fluid 36 hours after hCG administration; around this period, P4\nsecretion is considered to be unstable.  COX-2 and its metabolites are also very important\nbioactive substances in the formation of corpus lutea, though further study is required to clarify\nthe mechanism of how COX-2 and prostaglandins regulate this process. \nWe have demonstrated that COX-2 might not be produced at the primary follicle stage, but\nbegins to be produced at the secondary follicle stage in human ovary and continues to be\nexpressed in the granulosa cell layers of developing follicles until follicles reach the Graafian\nCOX-2 Expression during Follicular Development \n-45 -\n\nfollicle stage.  Just before the exposure to LH surge, COX-2 expression is temporarily\nwithdrawn.  We also showed that the existence of this enzyme in human follicular fluid after\nadministration of hCG in IVF-ET patients.  These findings collectively suggest that COX-2 is\nessential in follicular development and rupture in human ovary. \nAckowledgement\nWe thank the gynecologists at Osaka city medical School for their great support.  This Work\nwas supported by the Osaka Medical Research Foundation for Incurable Diseases. \nReferences\n1. Espey L L: Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory\nreaction. Biol Reprod 50: 233-238 (1994)\n2. Smith W L, DeWitt D L: Prostaglandin endoperixide H synthase-1 and -2. Adv Immunol 62: 167-215 (1996) \n3. Smith W L, Marnett L J: Prostaglandin endoperoxide synthase: structure and catalysis. Biochim Biophys\nActa 24: 1-17 (1991)\n4. Needleman P, Turk J, Jakschik B A, Morrison A R, Lefkowith J B: Arachidonic acid metabolism. Annu Rev\nBiochem 55: 69-102 (1986)\n5. Silva P J, Juengel J L, Rollyson M K, Niswender G D: Prostaglandin metabolism in the ovine corpus\nluteum: catabolism of prostaglandin F2\nα (PGF2α) coincides with resistance of the corpus luteum to PGF2 α .\nBiol Reprod 63: 1229-1236 (2000)\n6. 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Sirois J, Dore M: The late induction of prostaglandin G/H synthase-2 in equine preovulatory follicles\nsupports its role as a determinant of the ovulatory process. Endocrinology 138: 4427-4434 (1997)\n21. Duffy D M, Stouffer R L: The ovulatory gonadotrophin surge stimulates cyclooxygenase expression and\nprostaglandin production by the monkey follicle. Mol Hum Reprod 7: 731-739 (2001)\n22. Tokuyama O, Nakamura Y, Musoh A, Honda K, Ishiko O and Ogita S: Expression and distribution of\ncyclooxygenase-2 in human periovulatory ovary. Int J Mol Med 8: 603-606 (2001)\nCOX-2 Expression during Follicular Development \n-47 -","source_license":"CC0","license_restricted":false}