Vol. 33, pp , 2005 A FSH FSH. rfsh. rfsh 25 miu ml A rfsh 100 miu ml FSH FSH, TGF-b

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1 287 Vol. 33, pp , 2005 A : A A B FSH mrna FSH rfsh miuml rfsh A B rfsh rfsh 25 miuml A rfsh 100 miuml A B A FSH mrna A A A FSH mrna FSH FSH, FSH 1 2 FSH mrna FSH TGF-b a b 71

2 288 a ba A a bb B A 2 ba FSH FSH FSH FSH mrna 1 FSH FSH 2 a b a A A B FSH mrna 2 BDF1 C57BL6Lc slcdba2cr slc 2223C :0018 : C57Bl6 Lc slc CE-2 2 BDF1 a minimum essential medium eagle amem: SIGMA, USA 37C 5 CO 2 in air 30 G SIGMA, USA amem Dish Falcon : amem, 10 Fetal Bovine Serum FBS: 0.1 Insulintransferrin-sodium selenite ITS: SIGMA, USA 20 ml Oil for tissue culture, SAGE Biopharma, USA 5ml ml 1 DAY0 DAY1 10 ml 37C 5 CO 2 in air C 1 FSH FSH FSH rfsh: Follistim: Organon, Netherlands 25 miuml rfsh25 miuml 100 miuml rfsh100 miuml A B enzyme-linked immunosorbent assays ELISA 72

3 A A rfsh A SIGMA, USA A 100 ngml A B rfsh 25 miuml A 5 7 total RNA RT-PCR FSH mrna FSH mrna real-time PCR ELISA DAY DAY3 DAY11 1 Rodent Estradiol ELISA test Endocrine tech., USA solid-phase enzyme immunoassay based on competitive binding method pgml nm 5.0 pgml 2 A B A B ELISA Diagnostic System Lab. USA enzymatically amplified two-site two-step sandwich-type immunoassay 2 ELISA pgml nm A ELISA 1.0 pgml B ELISA 7.0 pgml 3 micro plate reader BioRad, JAPAN Apple Macintosh MPM III-Vs.1.57 FSH mrna 1 RNA RNAgents Total RNA Isolation System Promega, USA 5 7 PBS 2M g 4C 20 RNA 20C g 4C g 4C 10 total RNA 2 Reverse Transcriptase RT AMV Reverse Transcriptase XL for RT- PCR SmartSpec3000 BioRad RNA RT Total RNA 1 mg RNase Free H 2O, MgCl 2 RT bu#er, dntp mixture, RNase inhibitor, AMV reverse transcriptase, Random nona mers 20 ml 42C 30 RT 95C 5 RT 3 PCR Total RNA 0.1 mg cdna 20 mm primers, 5PCR bu#er, RNase Free H 2O, TaKaRa ExTaqHS 20 ml PCR Thermal cycler SP: PCR 2 ethidium bromide FSH primer 2001 Suresh Babu Table. 1 4 mrna real-time PCR real-time PCR Light Cycler Fast Start DNA Master SYBR Green I. Roche Diagnostics K.K., LD-DNA Master Syber Green, Mg Cl 2 18 ml RT 2 ml 73

4 z{ }~ 290 T Table 1. Oligonucleotide Primer Sequences Designed for PCR Amplification of FSH receptor and GAPDH ῌ real-time PCR ῌLight cy- cler: Roche Diagnostics K.K., PCR ῌ ῌ KL MN OPQRSTUV SAS Institute Inc. ῌUSA StatViewJ-5.0 (! +Y Z +Y[\ ]Z^!! "#$% &' _ ῌ "# 3 NdST`abeR fgfhij Fisher s Protected Least Significant "# 7 Nd*) RNA +, -. 2 / ῌ o pq Un- ST 11 Nd 34 ῌ : ;<=1 ῌmean S.E.M. Fig. 1 ῌ KL OPQR `abf 2ῐ3 c)g Di#erence ῌFisher s PLSD ) ῌ * paired Student s t-test WXQR*) `abfkcl mrnqri QRr fs "# 9 Nd QRt u(rnqrvi+y ῌ "# 11 NdST 13 Nd 3 > QRtFws WXQR)GH Graafian QR I- u uxy?@a 5 BC DE1FGHIJ ῌ ῌ Fig. 1. Follicle culture for 13 day. a.b. secondary follicle c. preantral follicle d.e. preantral follicle Granulosa cells have pierced through the basal membrane. f. antral follicle in which the antral-like cavity was formed. g. The antral-like cavity was expanded. h. Graafian follicle bars: 100 mm. 74

5 A 291 rfsh 1 rfsh Fig. 2 rfsh 25 miuml rfsh 100 miuml 5 7 rfsh 100 miu rfsh 25 miuml mm v.s mm,p mm v. s mm, p mm v.s mm, p mm v.s mm p mm v.s mm, p0.05 Fig. 2. Changes in diameters of follicles cultured for 13 days with 100 miuml or 25 miuml of rfsh. F100: rfsh 100 miuml n26 F25: rfsh 25 miuml n10 Diameters of follicles were significant larger in follicle cultured with rfsh 100 miuml. 2 rfsh 100 miuml rfsh 25 miuml Fig. 3 rfsh 100 miuml rfsh 25 miuml rfsh 100 miuml 3 A B rfsh A B Fig. 4 A rfsh 100 miuml 25 miuml ngml v.s ngml, p ngml v.s ngml, p ngml v.s ngml, p ngml v.s ngml, p0.05 B A rfsh 100 miuml ngml v.s ngml, p ngml v.s ngml, p0.05 rfsh 100 miuml 11 rfsh 25 Fig. 3. Survival rates of cultured follicles. F25: rfsh 25IUml n19 F100: rfsh 100 IUml n67 Survival rates were significant greater in follicles cultured with rfsh 100 miuml. 75

6 292 Fig. 4. Changes in inhibin A, B and estradiol concentrations in the medium. F25: rfsh 25 IUml n4 F100: rfsh 100 IUml n4 Concentrations of inhibin A, B and estradiol in the medium were increased significantly in follicles cultured with rfsh 100 miuml. miuml rfsh 100 miuml pgml v.s pgml, p pgml v.s pgml, p gml v.s pgml, p0.05 rfsh A 1 rfsh 25 miuml, rfsh 100 miuml rfsh A Fig. 5 rfsh 25 miuml A mm v.s mm, p mm v.s mm, p mm v.s mm, p mm v.s mm p mm v.s mm p0.01 A rfsh 25 miuml A rfsh 100 miuml rfsh 100 miuml A 2 rfsh A Fig. 6 rfsh 100 miuml A A rfsh 25 miuml A 3 A B rfsh A A B Fig. 7 76

7 A ngml, p0.05 rfsh 100 miuml A 11 A pg ml v. s pgml, p0.01 rfsh 25 miuml A A 4 A FSH mrna rfsh 25 miuml A 5 7 mrna FSH mrna real-time PCR Fig A FSH mrna v.s , p0.05 Fig. 5. Changes in diameters of follicles cultured for 13 days with rfsh only or with rfsh and activin A A F100: rfsh 100 miuml. n26 F100A: rfsh 100 miuml and activin A 100 ngml n35 B F25: rfsh 25 miuml. n10 F25A: rfsh 25 miuml and activin A 100 ngml n13 A A 11 A A 5 A ngml v.s ngml, p0.05 B A A ngml v.s Eppig Schroeder Eppig O Brien Cortvrindt 9 Graafian 77

8 294 Fig. 6. Survival rates of cultured follicles. F25 F: rfsh 25 IUml n19 FA : rfsh 25 IUml and activin A 100 ngml. n19 F100 F: rfsh 100 miuml and activin A 100 ngml n65 F A: rfsh 100 miuml and activin A 100 ngml n68 Survival rates significantly increased in follicles cultured with rfsh and activin A. Fig. 7. Changes in concentration of inhibin A, B and estradiol in medium. F25: rfsh 25 IUml n4 F25A: rfsh 25 IUml and activin A 100 ngml n4 F100: rfsh 100 miuml and activin A 100 ngml n4 F100A: rfsh 100 miuml and activin A 100 ngml n6 A InhibinA concentrations in the medium increased daily throughout the 11 days of culture, and tended to be promote further by addition of activina. B InhibinB concentrations in the medium did not change during the 11 day culture, however, addition of activina increased them significantly by day 11. C Estradiol concentrations in the medium did not change during the 9 day culture with rfsh 100 miuml. Addition of activina also did not influence estradiol levels until day 9 but decreased them significantly on day 11. Estradiol levels did not change time-dependently and were not influenced by addition of activina during 11day of culture with rfsh 25 miuml. 78

9 A 295 Fig. 8. Expression of FSH receptor mrna in 5 and 7 day follicles. Each group n6 follicles. Activin A promoted expression of FSH receptor mrna in 7day. Zheng 2 3 FSH in situ hybridization 10 Yamato FSH FSH 11 Sariram FSH 12 GnRH FSH hpghpg FSH 13 FSH 14 rfsh 57 rfsh rfsh FSH Schneyer A B 15 79

10 296 A B A rfsh 100 miuml 25 miuml A rfsh A B rfsh 100 miuml 25 miuml A B rfsh rfsh 100 miuml 11 rfsh 25 miuml rfsh A B 3 A B rfsh FSH 16 in vivo In vitro Zhao 19 Fiona 20 Mizumura FSH 21 2 rfsh 25 miuml 5 rfsh 100 miuml rfsh rfsh FSH, LH FSH LH A FSH mrna rfsh A 5 7 mrna 5 FSH mrna A 7 A FSH 15 A FSH mrna FSH mrna A FSH mrna FSH FSH A rfsh A A 80

11 A 297 A B A A A FSH mrna rfsh A A rfsh A FSH FSH FSH A FSH FSH A A FSH mrna rfsh Bio Science, 2001: : Yamoto M, Minami S and Nakano R. Immunohistochemical localization of inhibinactivin Subunits in human ovarian during the menstrual cycle. J Clin Endocrinol Metab 1992; 74: HORMONE FRONTIER IN GYNE- COLOGY 1998; 5, 3: Muttukrishna S, Fowler PA, George L, Groom NP and Knight PG. Changes in peripheral serum levels of total ActivinA during the Human menstrual cycle and pregnancy. J Clin Endocrinol Metab 1996; 81: Baker TG. Aquantitative and cytological study of germ cells in the human ovaries. Proc R Soc Lond Bio 1963; 153: Eppig JJ and Schroeder AC. Capacity of 81

12 298 mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilization in vitro. Biol Reprod 1989; 41: Eppig JJ and O Brien MJ. Comparison of preimplantation developmental competence after mouse growth and development in vitro and in vivo. Theriogenology 1998; 49: Cortvrindt R, Liu j and Smitz J. Validation of a simplified culture system for primary mouse follicles by birth of live young, In Ares Serono Symposia, XI-th litermational Workshop on Deveropment and Function of the Reproductive Organts. Artis Zoo, Amsterdam The Netherlands, Zheng W, Magid MS, Kramer EE and Chen YT. Follicle stimulation hormone receptor is expressed in human ovarian surface epithelium and fallopian tube. Am J Pathol 1996; 148: Yamato M, Shima K and Nakano R. gonadrotropin receptors in human ovarian follicles and corpora lutea throughout the menstrual cycles. Horm Res 1992; 37: Dierich A, Sairam MR, Monaco L, Fimia GM, Gansmuller A, LeMeur M and Sassone-Corsi P. Impairing Follicle stimulating hormone FSH signaling in vitro.: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance. Proc Natl Acd Sci USA 1998; 95: Halpin DM, Jones A, Fink G and Cjarlton HM. Postnatal ovarian follicle development in hypogonadal hpg and normal mice and associated changes in the hypothalamic pituitary ovarian axis. J Reprod Fertil 1986; 77: Oktay K, Newton H, Mullan J and Gosden RG. Development of human primodial follicles to antral stage in SCID hpg micestimulated with follicle stimulating hormone. Hum Reprod 1998; 13: Schneyer AL, Fujiwara T, Fox J, Welt CK, Adams J, Messerlian GM and Taylor AE. Dynamic changes in the intrafollicular inhibin activin follistatin axis during human follicular development : relationship to circulating hormone concentrations. J Clin Endocrinol Metab 2000; 85: ; 52: Doi M, Igarashi M, Hasegawa Y, Eto Y, Shibai H, Miura T and Ibuki Y. In vivo action of activin-a on pituitary-gonadal system. Endocrinology 1992; 130: Erickson GF, Kokka S and Rivier C. Activin causes premature superovulation. Endocrinology 1995; 136: Zhao J, Taverne MA, van der Weijden GC, Bevers MM and Van den Hurk R. E#ect of activina on in vitro development of rat preantral follicles and localization of activina and activin receptor II. Biol Reprod 2001; 65: Fiona HT, David GA and Evelyn ET. Activin promotes oocyte development in ovine preantral follicles in vitro. Reprod Biol and Endocrinol 2003; 1: Mizunuma H, Liu X, Andoh K, Abe Y, Kobayasi J, Yamada K, Yokota H, Ibuki Y and Hasegawa Y. Activin from secondary follicles causes small preantral follicles to remain dormant at the resting stage. Endocrinology 1999; 140: Xiao S, Robertson D and Findlay JK. E#ects of activin and follicle-stimulating-hormone FSH-suppressing proteinfollistatin on FSH receptor and Di#erentiation of cultured rat granurosa cells. Endocrinology 1992; 131: Kishi H, Minegishi T, Tano M, Kameda T, Ibuki Y and Miyomoto K, The e#ect of activin and FSH on the di#erentiation of rat gronulosa cell. FEBS Lett. 1998; 422: Minegishi T, Kishi H, Tano M, Kameda T, Hasegawa T and Miyamoto K, Control of FSH receptor mrna expression in rot granurosa cell by 3,5 cyclic adenosine monophosphate, activin and follistatin. Mol. Cell. End- 82

13 A 299 crinol 1999; 149: Miro F, Smyth CD and Hillier SG. Development-related e#ects of recombinant activin on steroid synthesis in rat granulosa cells. Endocrinology 1991; 177:

14 300 Abstract The E#ect of Activin A for the Cultured Follicle Growth Megumi Doi 1, Takahisa Mimuro 1, Eri Ishida 12, and Bunpei Ishizuka 1 It has been reported that activin A enhances follicle development in the ovary by an autocrine paracrine mechanism. This e#ect of activin A is reported to be di#erent according to developmental stage of the follicles. We established an immature mouse follicle culture system and investigated the e#ects of activin A on the growth and endocrine function of the follicles in vitro. For this purpose, we measured changes in follicle diameter, inhibin A, B and estradiol secretion, and FSH receptor mrna expression in the follicle in response to di#erent concentrations of rfsh 25 and 100 miuml with or without activin A. The follicular diameters were significantly enlarged by rfsh in dose dependent manner from preantral day 5 follicle stage. Inhibin A, B and the estradiol secretion from the follicles were significantly increased by rfsh in a dose related manner from the earlier preantral follicle stage day 3. Follicular diameters were significantly increased by addition of activin A to rfsh25 miuml. Inhibin A, B secretions were significantly increased by addition of activin A. Also the FSH receptor mrna expression in preantral follicle was significantly increased by activin A. The present study demonstrated that: i activin A enhances inhibin A, B secretion of early preantral follicle and then promotes enlargement of follicular diameter at the later preantral stage; ii activin A promotes FSH receptor mrna expression in earlier preantral stage follicles. These results suggest that activin A promotes follicular development at the transitional time from gonadotrophin independent to dependent phase through regulation of FSH receptor expression. key words follicle, FSH, activin, inhibin 1 Department of Obstetrics and Gynecology, St Marianna University School of Medicine 2 Laboratory of Cell Biology, Nihon University College of Bioresource Sciences 84