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PLOS ONE  2013 

Involvement of the Serine Protease Inhibitor, SERPINE2, and the Urokinase Plasminogen Activator in Cumulus Expansion and Oocyte Maturation

DOI: 10.1371/journal.pone.0074602

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The serpin peptidase inhibitor, clade E, member 2 (SERPINE2) inhibits urokinase-type plasminogen activator (PLAU) and tissue-type plasminogen activator. Higher SERPINE2 expression levels were detected in cumulus cells of human immature oocytes than in those of mature oocytes. The objective of this study was to evaluate whether high SERPINE2 levels in cumulus cells are associated with oocyte immaturity. Using the mouse cumulus–oocyte complex as an experimental model, the effects of elimination and overexpression of SERPINE2 in cumulus cells on cumulus expansion and oocyte maturation were assayed by in vitro maturation. Serpine2 and PLAU transcripts were the most highly expressed serpins and plasminogen activators, respectively. Their expression was coordinately regulated in cumulus cells during gonadotropin-induced oocyte maturation. Silencing of Serpine2 expression using small interfering RNAs or blockage of SERPINE2 protein using a specific antibody had no effect on oocyte maturation. However, overexpression of Serpine2 or exogenous supplementation with high levels of SERPINE2 impaired cumulus expansion and oocyte maturation, probably by decreasing hyaluronan synthase 2 (Has2) and versican (Vcan) mRNA expression. Amiloride, a specific PLAU inhibitor, also suppressed these processes. PLAU supplementation of the oocyte in vitro maturation medium caused earlier and more extensive expansion of cumulus cells and oocyte maturation that may be mediated by increased Has2 mRNA expression. However, these effects were neutralized by coincubation with SERPINE2 or amiloride and PLAU. In conclusion, SERPINE2 and PLAU are involved in cumulus expansion and oocyte maturation. High SERPINE2 levels impair these processes, probably by decreasing cumulus matrix gene expression as well as reducing cumulus hyaluronan contents and inhibiting PLAU activity. These findings may explain why cumulus cells surrounding immature human oocytes express high SERPINE2 levels.


[1]  Zhuo L, Yoneda M, Zhao M, Yingsung W, Yoshida N, et al. (2001) Defect in SHAP-hyaluronan complex causes severe female infertility. A study by inactivation of the bikunin gene in mice. J Biol Chem 276: 7693–7696.
[2]  Salustri A, Garlanda C, Hirsch E, De Acetis M, Maccagno A, et al. (2004) PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization. Development 131: 1577–1586.
[3]  Varani S, Elvin JA, Yan C, DeMayo J, DeMayo FJ, et al. (2002) Knockout of pentraxin 3, a downstream target of growth differentiation factor-9, causes female subfertility. Mol Endocrinol 16: 1154–1167.
[4]  Fulop C, Szanto S, Mukhopadhyay D, Bardos T, Kamath RV, et al. (2003) Impaired cumulus mucification and female sterility in tumor necrosis factor-induced protein-6 deficient mice. Development 130: 2253–2261.
[5]  Brown HM, Dunning KR, Robker RL, Boerboom D, Pritchard M, et al. (2010) ADAMTS1 cleavage of versican mediates essential structural remodeling of the ovarian follicle and cumulus-oocyte matrix during ovulation in mice. Biol Reprod 83: 549–557.
[6]  Rugg MS, Willis AC, Mukhopadhyay D, Hascall VC, Fries E, et al. (2005) Characterization of complexes formed between TSG-6 and inter-alpha-inhibitor that act as intermediates in the covalent transfer of heavy chains onto hyaluronan. J Biol Chem 280: 25674–25686.
[7]  Scarchilli L, Camaioni A, Bottazzi B, Negri V, Doni A, et al. (2007) PTX3 interacts with inter-alpha-trypsin inhibitor: implications for hyaluronan organization and cumulus oophorus expansion. J Biol Chem 282: 30161–30170.
[8]  Yokoo M, Sato E (2004) Cumulus-oocyte complex interactions during oocyte maturation. Int Rev Cytol 235: 251–291.
[9]  Gui LM, Joyce IM (2005) RNA interference evidence that growth differentiation factor-9 mediates oocyte regulation of cumulus expansion in mice. Biol Reprod 72: 195–199.
[10]  Su YQ, Wu X, O'Brien MJ, Pendola FL, Denegre JN, et al. (2004) Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop. Dev Biol 276: 64–73.
[11]  Eppig JJ (2001) Oocyte control of ovarian follicular development and function in mammals. Reproduction 122: 829–838.
[12]  Matzuk MM, Burns KH, Viveiros MM, Eppig JJ (2002) Intercellular communication in the mammalian ovary: oocytes carry the conversation. Science 296: 2178–2180.
[13]  D'Alessandris C, Canipari R, Di Giacomo M, Epifano O, Camaioni A, et al. (2001) Control of mouse cumulus cell-oocyte complex integrity before and after ovulation: plasminogen activator synthesis and matrix degradation. Endocrinology 142: 3033–3040.
[14]  Leonardsson G, Peng XR, Liu K, Nordstrom L, Carmeliet P, et al. (1995) Ovulation efficiency is reduced in mice that lack plasminogen activator gene function: functional redundancy among physiological plasminogen activators. Proc Natl Acad Sci U S A 92: 12446–12450.
[15]  Peng XR, Hsueh AJ, Ny T (1993) Transient and cell-specific expression of tissue-type plasminogen activator and plasminogen-activator-inhibitor type 1 results in controlled and directed proteolysis during gonadotropin-induced ovulation. Eur J Biochem 214: 147–156.
[16]  Tsafriri A, Bicsak TA, Cajander SB, Ny T, Hsueh AJ (1989) Suppression of ovulation rate by antibodies to tissue-type plasminogen activator and alpha 2-antiplasmin. Endocrinology 124: 415–421.
[17]  Yoshimura Y, Santulli R, Atlas SJ, Fujii S, Wallach EE (1987) The effects of proteolytic enzymes on in vitro ovulation in the rabbit. Am J Obstet Gynecol 157: 468–475.
[18]  Sappino AP, Huarte J, Vassalli JD, Belin D (1991) Sites of synthesis of urokinase and tissue-type plasminogen activators in the murine kidney. J Clin Invest 87: 962–970.
[19]  Hofmann GE, Glatstein I, Schatz F, Heller D, Deligdisch L (1994) Immunohistochemical localization of urokinase-type plasminogen activator and the plasminogen activator inhibitors 1 and 2 in early human implantation sites. Am J Obstet Gynecol 170: 671–676.
[20]  Vassalli JD, Sappino AP, Belin D (1991) The plasminogen activator/plasmin system. J Clin Invest 88: 1067–1072.
[21]  Scott RW, Bergman BL, Bajpai A, Hersh RT, Rodriguez H, et al. (1985) Protease nexin. Properties and a modified purification procedure. J Biol Chem 260: 7029–7034.
[22]  Chen LM, Zhang X, Chai KX (2004) Regulation of prostasin expression and function in the prostate. Prostate 59: 1–12.
[23]  Chern SR, Li SH, Chiu CL, Chang HH, Chen CP, et al. (2011) Spatiotemporal expression of SERPINE2 in the human placenta and its role in extravillous trophoblast migration and invasion. Reprod Biol Endocrinol 9: 106.
[24]  Chern SR, Li SH, Lu CH, Chen EI (2010) Spatiotemporal expression of the serine protease inhibitor, SERPINE2, in the mouse placenta and uterus during the estrous cycle, pregnancy, and lactation. Reprod Biol Endocrinol 8: 127.
[25]  Lee RK, Fan CC, Hwu YM, Lu CH, Lin MH, et al. (2011) SERPINE2, an inhibitor of plasminogen activators, is highly expressed in the human endometrium during the secretory phase. Reprod Biol Endocrinol 9: 38.
[26]  Bedard J, Brule S, Price CA, Silversides DW, Lussier JG (2003) Serine protease inhibitor-E2 (SERPINE2) is differentially expressed in granulosa cells of dominant follicle in cattle. Mol Reprod Dev 64: 152–165.
[27]  Hagglund AC, Ny A, Liu K, Ny T (1996) Coordinated and cell-specific induction of both physiological plasminogen activators creates functionally redundant mechanisms for plasmin formation during ovulation. Endocrinology 137: 5671–5677.
[28]  Vassalli JD, Huarte J, Bosco D, Sappino AP, Sappino N, et al. (1993) Protease-nexin I as an androgen-dependent secretory product of the murine seminal vesicle. EMBO J 12: 1871–1878.
[29]  Lu CH, Lee RK, Hwu YM, Chu SL, Chen YJ, et al. (2011) SERPINE2, a serine protease inhibitor extensively expressed in adult male mouse reproductive tissues, may serve as a murine sperm decapacitation factor. Biol Reprod 84: 514–525.
[30]  Liu YX, Hsueh AJ (1987) Plasminogen activator activity in cumulus-oocyte complexes of gonadotropin-treated rats during the periovulatory period. Biol Reprod 36: 1055–1062.
[31]  Liu YX, Liu K, Feng Q, Hu ZY, Liu HZ, et al. (2004) Tissue-type plasminogen activator and its inhibitor plasminogen activator inhibitor type 1 are coordinately expressed during ovulation in the rhesus monkey. Endocrinology 145: 1767–1775.
[32]  Liu K, Wahlberg P, Leonardsson G, Hagglund AC, Ny A, et al. (2006) Successful ovulation in plasminogen-deficient mice treated with the broad-spectrum matrix metalloproteinase inhibitor galardin. Dev Biol 295: 615–622.
[33]  Devjak R, Fon Tacer K, Juvan P, Virant Klun I, Rozman D, et al. (2012) Cumulus cells gene expression profiling in terms of oocyte maturity in controlled ovarian hyperstimulation using GnRH agonist or GnRH antagonist. PLoS One 7: e47106.
[34]  Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408.
[35]  Dragovic RA, Ritter LJ, Schulz SJ, Amato F, Thompson JG, et al. (2007) Oocyte-secreted factor activation of SMAD 2/3 signaling enables initiation of mouse cumulus cell expansion. Biol Reprod 76: 848–857.
[36]  Mahmodi R, Abbasi M, Amiri I, Kashani IR, Pasbakhsh P, et al. (2009) Cumulus cell role on mouse germinal vesicle oocyte maturation, fertilization, and subsequent embryo development to blastocyst stage in vitro. Yakhteh Medical Journal 11: 299–302.
[37]  Jankun J, Skrzypczak-Jankun E (1999) Molecular basis of specific inhibition of urokinase plasminogen activator by amiloride. Cancer Biochem Biophys 17: 109–123.
[38]  Weigel PH, Hascall VC, Tammi M (1997) Hyaluronan synthases. J Biol Chem 272: 13997–14000.
[39]  Cao M, Nicola E, Portela VM, Price CA (2006) Regulation of serine protease inhibitor-E2 and plasminogen activator expression and secretion by follicle stimulating hormone and growth factors in non-luteinizing bovine granulosa cells in vitro. Matrix Biol 25: 342–354.
[40]  Dunning KR, Lane M, Brown HM, Yeo C, Robker RL, et al. (2007) Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes. Hum Reprod 22: 2842–2850.
[41]  Murer V, Spetz JF, Hengst U, Altrogge LM, de Agostini A, et al. (2001) Male fertility defects in mice lacking the serine protease inhibitor protease nexin-1. Proc Natl Acad Sci U S A 98: 3029–3033.
[42]  Cao M, Sahmi M, Lussier JG, Price CA (2004) Plasminogen activator and serine protease inhibitor-E2 (protease nexin-1) expression by bovine granulosa cells in vitro. Biol Reprod 71: 887–893.
[43]  Ny A, Nordstrom L, Carmeliet P, Ny T (1997) Studies of mice lacking plasminogen activator gene function suggest that plasmin production prior to ovulation exceeds the amount needed for optimal ovulation efficiency. Eur J Biochem 244: 487–493.
[44]  Richards JS (2005) Ovulation: new factors that prepare the oocyte for fertilization. Mol Cell Endocrinol 234: 75–79.
[45]  Cha KY, Chian RC (1998) Maturation in vitro of immature human oocytes for clinical use. Hum Reprod Update 4: 103–120.
[46]  Trounson A, Anderiesz C, Jones G (2001) Maturation of human oocytes in vitro and their developmental competence. Reproduction 121: 51–75.


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