Animal Biotechnology
ISSN: 1049-5398 (Print) 1532-2378 (Online) Journal homepage: http://www.tandfonline.com/loi/labt20
Formation of an Antral Follicle-Like Structure by Bovine Cumulus-Oocyte Complexes Embedded with Fragmin/Protamine Microparticles Yasuhiro Morita, Ni Wayan Kurniani Karja, Lanh Thi Kim Do, Fuminori Tanihara, Masayasu Taniguchi & Takeshige Otoi To cite this article: Yasuhiro Morita, Ni Wayan Kurniani Karja, Lanh Thi Kim Do, Fuminori Tanihara, Masayasu Taniguchi & Takeshige Otoi (2015) Formation of an Antral FollicleLike Structure by Bovine Cumulus-Oocyte Complexes Embedded with Fragmin/Protamine Microparticles, Animal Biotechnology, 26:4, 273-275 To link to this article: http://dx.doi.org/10.1080/10495398.2015.1015681
Published online: 09 Jul 2015.
Submit your article to this journal
Article views: 31
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=labt20 Download by: [University of Nebraska, Lincoln]
Date: 04 October 2015, At: 21:06
Animal Biotechnology, 26:273–275, 2015 Copyright # Taylor & Francis Group, LLC ISSN: 1049-5398 print=1532-2378 online DOI: 10.1080/10495398.2015.1015681
Formation of an Antral Follicle-Like Structure by Bovine Cumulus-Oocyte Complexes Embedded with Fragmin/ Protamine Microparticles Yasuhiro Morita,1 Ni Wayan Kurniani Karja,2 Lanh Thi Kim Do,1 Fuminori Tanihara,1 Masayasu Taniguchi,1 and Takeshige Otoi1 1
Downloaded by [University of Nebraska, Lincoln] at 21:06 04 October 2015
Laboratory of Animal Reproduction, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan 2 Division of Reproduction, Department of Clinic, Reproduction, and Pathology, Bogor Agriculture University, Indonesia Fragmin/protamine microparticles (F/P MPs) approximately 0.5-1 lM in diameter were prepared by the simple mixing of fragmin with protamine. This study investigated the effects of F=P MP-containing collagen gels as a hormone carrier on the formation of antral follicle-like structures and on the development of growing bovine oocytes. The supplementation of F=P MPs in collagen gels contributed to the beneficial effects of follicle stimulating hormone (FSH) on the formation and size of antral follicle-like structures. The F=P MPs may serve as potential hormone carriers for the growth of cultured bovine oocytes from early antral follicles. Keywords
Follicular formation; FSH; In vitro culture; Nanoparticles; Oocyte
INTRODUCTION In vitro growth culture techniques for small oocytes not only provide a novel source of mature oocytes for livestock production but also have become an important model for understanding the mechanisms of follicle growth and oocyte maturation. Three-dimensional culture systems have been developed to support the growth of small oocytes in mouse (1), cow (2), and pig models (3). Generally, the use of a collagen gel matrix helps to prevent migration of cumulus cells away from oocytes and also supports a spherical structure of follicles (4). Fragmin=protamine microparticles (F=P MPs) have been used as carriers for the preservation and controlled release of growth factors and to alter cell viscosity and adherence (5, 6). In the present study, we investigated the supplementation effects of F=P MP in the collagen gels as a hormone carrier on the formation of an antral follicle-like structure (AFLS) and on the development of growing bovine oocytes.
MATERIALS AND METHODS The preparation of F=P MPs was performed according to the method described by Nakamura et al. (7). Briefly, Address correspondence to Takeshige Otoi, Laboratory of Animal Reproduction, The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1, Yoshida, Yamaguchi 753-8515, Japan. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/labt.
0.3 mL of a protamine solution (Mochida Pharmaceutical Co.) was added dropwise to 0.7 mL of a fragmin solution (Kissei Pharmaceutical Co.), and then, the solution was vortexed to produce microparticles. The solution of F=P MPs was resuspended in 1 mL phosphate-buffered saline (PBS, Invitrogen, Carlsbad, CA, USA). Oocytes-cumulus complexes with pieces of parietal granulosa (OCCGs) were dissected from follicles that were 0.5 to 0.7 mM in diameter (Fig. 1a). The OCCGs containing oocytes 85 to 97 mM in diameter were embedded in a collagen gel according to a previously reported method (8). The embedded OCCGs were cultured in 600 mL of growth medium (TCMl99 medium with Earle’s salts buffered with 25 mM HEPES buffer and supplemented with 10% fetal bovine serum, 50 mg=mL gentamicin, and 4 mM hypoxanthine) at 38.5 C in 5% CO2 and 95% air for 8 days. To evaluate the supplementation effects of F=P MPs and follicle stimulating hormone (FSH) (Kawasaki Seiyaku K.K.) on AFLS formation (Fig. 1b and c) and the growth of oocytes, the OCCGs were embedded in collagen gels supplemented with 10% (v=v) F=P MPs (MPs group), 10% (v=v) PBS containing 1.0 AU=mL of FSH (final concentration of 0.1 AU=mL) (FSH group), or 10% F=P MPs containing 1.0 AU=mL FSH (MPs=FSH group). As control, the OCCGs were embedded in collagen gels supplemented with 10% (v=v) PBS without MPs or FSH. The diameters of the AFLSs were measured 4 and 8 days after the start of culture. The diameters of oocytes with AFLS formation were measured before and after growth
273
274
Y. MORITA ET AL.
Downloaded by [University of Nebraska, Lincoln] at 21:06 04 October 2015
FIG. 1. Morphology of an antral follicle-like structure (AFLS) from bovine cumulus–oocyte complexes (a) embedded in collagen gel. The complexes were cultured for 4 days (b) and 8 days (c). The cumulus=granulosa cells spread into the collagen gel from 4 days after the start of culture. Scale bar represents 100 mm. culture for 8 days. The meiotic stage in the oocytes were analyzed by staining with the DNA-binding fluorochrome bisbenzimide (Hoechst 33342) that was previously described by Abakushina et al. (9). The AFLS formation rates, AFLS diameters, and oocyte diameters were tested by analysis of variance with a subsequent Fisher’s protected least significant difference (PLSD) test. The differences with a probability value (p) of 0.05 or less were considered significant. RESULTS AND DISCUSSION The rates of AFLS formation after 4 days of growth culture were generally higher (p < 0.1) in the MPs=FSH group than in the control group (Fig. 2). The mean diameters of the AFLSs after 4 and 8 days of growth culture were significantly higher (p < 0.05) in the MPs=FSH groups than in control groups (Fig. 3). After 8 days of growth culture, the mean diameters of oocytes with AFLS formation increased from 91.4 0.5 mm to 116.2 1.3 mm, but no
FIG. 2. Antral follicle-like structure (AFLS) formation by bovine oocytes-cumulus complexes with pieces of parietal granulosa (OCCGs) after culture for 4 and 8 days. The OCCGs were embedded in collagen gels (C), collagen gels containing 0.1 AU=mL FSH (F), collagen gels containing 10% MPs (M), or collagen gels containing 10% MPs and 0.1 AU=mL FSH (MF). Four to 5 replicated trials were carried out. The numbers within parentheses indicate the total number of OCCGs examined. Each bar represents the mean value SEM. Different from the control group (p < 0.1).
FIG. 3. Mean diameter of the antral follicle-like structure (AFLS) of bovine oocytes-cumulus complexes with pieces of parietal granulosa (OCCGs) after culture for 4 days and 8 days. The OCCGs were embedded in collagen gels (C), collagen gels containing 0.1 AU=mL FSH (F), collagen gels containing 10% MPs (M), or collagen gels containing 10% MPs and 0.1 AU=mL FSH (MF). The numbers within parentheses indicate the total number of AFLS examined. Each bar represents the mean value SEM. The bars with disparate letters for each time point differ significantly (p < 0.05).
significant differences in the increase of oocyte diameter were observed among the groups (23.7 to 28.2 mm). Moreover, more than 50% of the oocytes resumed meiosis, but none of the oocytes reached the metaphase II (MII) stage. In the present study, we observed that the supplementation of both F=P MPs and FSH improved the rate of AFLS formation after 4 days of growth culture and the mean diameters of AFLS after 8 days of culture. In contrast, the supplementation of FSH alone improved the diameters of AFLS after 4 days of culture but had no effects on the rate of AFLS formation. FSH has been suggested to stimulate the proliferation of bovine granulosa cells and maintain morphological quality, consequently allowing the formation of antrum-like structures (2). In the present study, FSH was supplemented in collagen gels but not in growth culture medium. Therefore, our results indicate that the supplementation of F=P MPs as a hormone carrier might contribute to FSH’s beneficial effects on the proliferation of bovine granulosa cells, resulting in an increase in the formation and diameter of the AFLSs. In the present study, we did not observe a beneficial effect from F=P MP supplementation on the diameter of oocytes after growth culture. The mechanisms underlying the effects of F=P MP supplementation are difficult to discern, but one possible explanation is that, as a hormone carrier, the F=P MPs only provide a mechanical support for the formation of AFLS and do not directly regulate the growth of oocytes (10). In conclusion, the results of this study demonstrate that F=P MPs with FSH promote the formation and increase in size of AFLSs but not the growth of oocytes. The F=P MPs may serve as potential hormone carriers for the growth culture of bovine oocytes from early antral follicles.
FOLLICULAR FORMATION OF OOCYTES WITH MICROPARTICLES
Downloaded by [University of Nebraska, Lincoln] at 21:06 04 October 2015
REFERENCES 1. Eppig JJ, Schroeder AC. Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilization in vitro. Biol Reprod 1989; 41:268–276. 2. Harada M, Miyano T, Matsumura K, Osaki S, Miyake M, Kato S. Bovine oocytes from early antral follicles grow to meiotic competence in vitro: effect of FSH and hypoxanthine. Theriogenology 1997; 48:743–755. 3. Hirao Y, Nagai T, Kubo M, Miyano T, Miyake M, Kato S. In vitro growth and maturation of pig oocytes. J Reprod Fertil 1994; 100:333–339. 4. Carroll J, Whittingham DG, Wood MJ. Effect of dibutyryl cyclic adenosine monophosphate on granulosa cell proliferation, oocyte growth and meiotic maturation in isolated mouse primary ovarian follicles cultured in collagen gels. J Reprod Fertil 1991; 92:197–207. 5. Yeo JE, Nam BM, Yang W, Jo YH, Lee S, Nemeno JG, Kiml BY, Koh YG, Lee JI. Fragmin=protamine microparticle carriers as a drug repositioning strategy for cell transplantation. Transplant Proc 2013; 45:3122–3126. 6. Kishimoto S, Ishihara M, Mori Y, Takikawa M, Sumi Y, Nakamura S, Sato T, Kiyosawa T. Three-dimensional
7.
8.
9.
10.
275
expansion using plasma-medium gel with fragmin=protamine nanoparticles and fgf-2 to stimulate adipose-derived stromal cells and bone marrow-derived mesenchymal stem cells. Biores Open Access 2012; 1:314–323. Nakamura S, Kanatani Y, Kishimoto S, Ohno C, Horio T, Masanori F, Hattori H, Tanaka Y, Kiyosawa T, Maehara T, Ishihara M. Controlled release of FGF-2 using fragmin=protamine microparticles and effect on neovascularization. J Biomed Mater Res A 2009; 91: 814–823. Yamamoto K, Otoi T, Koyama N, Horikita N, Tachikawa S, Miyano T. Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro. Theriogenology 1999; 52:81–89. Abakushina EV, Morita Y, Kaedei Y, Tanihara F, Namula Z, Viet VL, Otoi T. Formation of an antral follicle-like structure of bovine cumulus-oocyte complexes embedded individually or in groups in collagen gels. Reprod Domest Anim 2011; 46:423–427. Kreeger PK, Deck JW, Woodruff TK, Shea LD. The in vitro regulation of ovarian follicle development using alginate-extracellular matrix gels. Biomaterials 2006; 27: 714–723.
ISSN: 1049-5398 (Print) 1532-2378 (Online) Journal homepage: http://www.tandfonline.com/loi/labt20
Formation of an Antral Follicle-Like Structure by Bovine Cumulus-Oocyte Complexes Embedded with Fragmin/Protamine Microparticles Yasuhiro Morita, Ni Wayan Kurniani Karja, Lanh Thi Kim Do, Fuminori Tanihara, Masayasu Taniguchi & Takeshige Otoi To cite this article: Yasuhiro Morita, Ni Wayan Kurniani Karja, Lanh Thi Kim Do, Fuminori Tanihara, Masayasu Taniguchi & Takeshige Otoi (2015) Formation of an Antral FollicleLike Structure by Bovine Cumulus-Oocyte Complexes Embedded with Fragmin/Protamine Microparticles, Animal Biotechnology, 26:4, 273-275 To link to this article: http://dx.doi.org/10.1080/10495398.2015.1015681
Published online: 09 Jul 2015.
Submit your article to this journal
Article views: 31
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=labt20 Download by: [University of Nebraska, Lincoln]
Date: 04 October 2015, At: 21:06
Animal Biotechnology, 26:273–275, 2015 Copyright # Taylor & Francis Group, LLC ISSN: 1049-5398 print=1532-2378 online DOI: 10.1080/10495398.2015.1015681
Formation of an Antral Follicle-Like Structure by Bovine Cumulus-Oocyte Complexes Embedded with Fragmin/ Protamine Microparticles Yasuhiro Morita,1 Ni Wayan Kurniani Karja,2 Lanh Thi Kim Do,1 Fuminori Tanihara,1 Masayasu Taniguchi,1 and Takeshige Otoi1 1
Downloaded by [University of Nebraska, Lincoln] at 21:06 04 October 2015
Laboratory of Animal Reproduction, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan 2 Division of Reproduction, Department of Clinic, Reproduction, and Pathology, Bogor Agriculture University, Indonesia Fragmin/protamine microparticles (F/P MPs) approximately 0.5-1 lM in diameter were prepared by the simple mixing of fragmin with protamine. This study investigated the effects of F=P MP-containing collagen gels as a hormone carrier on the formation of antral follicle-like structures and on the development of growing bovine oocytes. The supplementation of F=P MPs in collagen gels contributed to the beneficial effects of follicle stimulating hormone (FSH) on the formation and size of antral follicle-like structures. The F=P MPs may serve as potential hormone carriers for the growth of cultured bovine oocytes from early antral follicles. Keywords
Follicular formation; FSH; In vitro culture; Nanoparticles; Oocyte
INTRODUCTION In vitro growth culture techniques for small oocytes not only provide a novel source of mature oocytes for livestock production but also have become an important model for understanding the mechanisms of follicle growth and oocyte maturation. Three-dimensional culture systems have been developed to support the growth of small oocytes in mouse (1), cow (2), and pig models (3). Generally, the use of a collagen gel matrix helps to prevent migration of cumulus cells away from oocytes and also supports a spherical structure of follicles (4). Fragmin=protamine microparticles (F=P MPs) have been used as carriers for the preservation and controlled release of growth factors and to alter cell viscosity and adherence (5, 6). In the present study, we investigated the supplementation effects of F=P MP in the collagen gels as a hormone carrier on the formation of an antral follicle-like structure (AFLS) and on the development of growing bovine oocytes.
MATERIALS AND METHODS The preparation of F=P MPs was performed according to the method described by Nakamura et al. (7). Briefly, Address correspondence to Takeshige Otoi, Laboratory of Animal Reproduction, The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1, Yoshida, Yamaguchi 753-8515, Japan. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/labt.
0.3 mL of a protamine solution (Mochida Pharmaceutical Co.) was added dropwise to 0.7 mL of a fragmin solution (Kissei Pharmaceutical Co.), and then, the solution was vortexed to produce microparticles. The solution of F=P MPs was resuspended in 1 mL phosphate-buffered saline (PBS, Invitrogen, Carlsbad, CA, USA). Oocytes-cumulus complexes with pieces of parietal granulosa (OCCGs) were dissected from follicles that were 0.5 to 0.7 mM in diameter (Fig. 1a). The OCCGs containing oocytes 85 to 97 mM in diameter were embedded in a collagen gel according to a previously reported method (8). The embedded OCCGs were cultured in 600 mL of growth medium (TCMl99 medium with Earle’s salts buffered with 25 mM HEPES buffer and supplemented with 10% fetal bovine serum, 50 mg=mL gentamicin, and 4 mM hypoxanthine) at 38.5 C in 5% CO2 and 95% air for 8 days. To evaluate the supplementation effects of F=P MPs and follicle stimulating hormone (FSH) (Kawasaki Seiyaku K.K.) on AFLS formation (Fig. 1b and c) and the growth of oocytes, the OCCGs were embedded in collagen gels supplemented with 10% (v=v) F=P MPs (MPs group), 10% (v=v) PBS containing 1.0 AU=mL of FSH (final concentration of 0.1 AU=mL) (FSH group), or 10% F=P MPs containing 1.0 AU=mL FSH (MPs=FSH group). As control, the OCCGs were embedded in collagen gels supplemented with 10% (v=v) PBS without MPs or FSH. The diameters of the AFLSs were measured 4 and 8 days after the start of culture. The diameters of oocytes with AFLS formation were measured before and after growth
273
274
Y. MORITA ET AL.
Downloaded by [University of Nebraska, Lincoln] at 21:06 04 October 2015
FIG. 1. Morphology of an antral follicle-like structure (AFLS) from bovine cumulus–oocyte complexes (a) embedded in collagen gel. The complexes were cultured for 4 days (b) and 8 days (c). The cumulus=granulosa cells spread into the collagen gel from 4 days after the start of culture. Scale bar represents 100 mm. culture for 8 days. The meiotic stage in the oocytes were analyzed by staining with the DNA-binding fluorochrome bisbenzimide (Hoechst 33342) that was previously described by Abakushina et al. (9). The AFLS formation rates, AFLS diameters, and oocyte diameters were tested by analysis of variance with a subsequent Fisher’s protected least significant difference (PLSD) test. The differences with a probability value (p) of 0.05 or less were considered significant. RESULTS AND DISCUSSION The rates of AFLS formation after 4 days of growth culture were generally higher (p < 0.1) in the MPs=FSH group than in the control group (Fig. 2). The mean diameters of the AFLSs after 4 and 8 days of growth culture were significantly higher (p < 0.05) in the MPs=FSH groups than in control groups (Fig. 3). After 8 days of growth culture, the mean diameters of oocytes with AFLS formation increased from 91.4 0.5 mm to 116.2 1.3 mm, but no
FIG. 2. Antral follicle-like structure (AFLS) formation by bovine oocytes-cumulus complexes with pieces of parietal granulosa (OCCGs) after culture for 4 and 8 days. The OCCGs were embedded in collagen gels (C), collagen gels containing 0.1 AU=mL FSH (F), collagen gels containing 10% MPs (M), or collagen gels containing 10% MPs and 0.1 AU=mL FSH (MF). Four to 5 replicated trials were carried out. The numbers within parentheses indicate the total number of OCCGs examined. Each bar represents the mean value SEM. Different from the control group (p < 0.1).
FIG. 3. Mean diameter of the antral follicle-like structure (AFLS) of bovine oocytes-cumulus complexes with pieces of parietal granulosa (OCCGs) after culture for 4 days and 8 days. The OCCGs were embedded in collagen gels (C), collagen gels containing 0.1 AU=mL FSH (F), collagen gels containing 10% MPs (M), or collagen gels containing 10% MPs and 0.1 AU=mL FSH (MF). The numbers within parentheses indicate the total number of AFLS examined. Each bar represents the mean value SEM. The bars with disparate letters for each time point differ significantly (p < 0.05).
significant differences in the increase of oocyte diameter were observed among the groups (23.7 to 28.2 mm). Moreover, more than 50% of the oocytes resumed meiosis, but none of the oocytes reached the metaphase II (MII) stage. In the present study, we observed that the supplementation of both F=P MPs and FSH improved the rate of AFLS formation after 4 days of growth culture and the mean diameters of AFLS after 8 days of culture. In contrast, the supplementation of FSH alone improved the diameters of AFLS after 4 days of culture but had no effects on the rate of AFLS formation. FSH has been suggested to stimulate the proliferation of bovine granulosa cells and maintain morphological quality, consequently allowing the formation of antrum-like structures (2). In the present study, FSH was supplemented in collagen gels but not in growth culture medium. Therefore, our results indicate that the supplementation of F=P MPs as a hormone carrier might contribute to FSH’s beneficial effects on the proliferation of bovine granulosa cells, resulting in an increase in the formation and diameter of the AFLSs. In the present study, we did not observe a beneficial effect from F=P MP supplementation on the diameter of oocytes after growth culture. The mechanisms underlying the effects of F=P MP supplementation are difficult to discern, but one possible explanation is that, as a hormone carrier, the F=P MPs only provide a mechanical support for the formation of AFLS and do not directly regulate the growth of oocytes (10). In conclusion, the results of this study demonstrate that F=P MPs with FSH promote the formation and increase in size of AFLSs but not the growth of oocytes. The F=P MPs may serve as potential hormone carriers for the growth culture of bovine oocytes from early antral follicles.
FOLLICULAR FORMATION OF OOCYTES WITH MICROPARTICLES
Downloaded by [University of Nebraska, Lincoln] at 21:06 04 October 2015
REFERENCES 1. Eppig JJ, Schroeder AC. Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilization in vitro. Biol Reprod 1989; 41:268–276. 2. Harada M, Miyano T, Matsumura K, Osaki S, Miyake M, Kato S. Bovine oocytes from early antral follicles grow to meiotic competence in vitro: effect of FSH and hypoxanthine. Theriogenology 1997; 48:743–755. 3. Hirao Y, Nagai T, Kubo M, Miyano T, Miyake M, Kato S. In vitro growth and maturation of pig oocytes. J Reprod Fertil 1994; 100:333–339. 4. Carroll J, Whittingham DG, Wood MJ. Effect of dibutyryl cyclic adenosine monophosphate on granulosa cell proliferation, oocyte growth and meiotic maturation in isolated mouse primary ovarian follicles cultured in collagen gels. J Reprod Fertil 1991; 92:197–207. 5. Yeo JE, Nam BM, Yang W, Jo YH, Lee S, Nemeno JG, Kiml BY, Koh YG, Lee JI. Fragmin=protamine microparticle carriers as a drug repositioning strategy for cell transplantation. Transplant Proc 2013; 45:3122–3126. 6. Kishimoto S, Ishihara M, Mori Y, Takikawa M, Sumi Y, Nakamura S, Sato T, Kiyosawa T. Three-dimensional
7.
8.
9.
10.
275
expansion using plasma-medium gel with fragmin=protamine nanoparticles and fgf-2 to stimulate adipose-derived stromal cells and bone marrow-derived mesenchymal stem cells. Biores Open Access 2012; 1:314–323. Nakamura S, Kanatani Y, Kishimoto S, Ohno C, Horio T, Masanori F, Hattori H, Tanaka Y, Kiyosawa T, Maehara T, Ishihara M. Controlled release of FGF-2 using fragmin=protamine microparticles and effect on neovascularization. J Biomed Mater Res A 2009; 91: 814–823. Yamamoto K, Otoi T, Koyama N, Horikita N, Tachikawa S, Miyano T. Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro. Theriogenology 1999; 52:81–89. Abakushina EV, Morita Y, Kaedei Y, Tanihara F, Namula Z, Viet VL, Otoi T. Formation of an antral follicle-like structure of bovine cumulus-oocyte complexes embedded individually or in groups in collagen gels. Reprod Domest Anim 2011; 46:423–427. Kreeger PK, Deck JW, Woodruff TK, Shea LD. The in vitro regulation of ovarian follicle development using alginate-extracellular matrix gels. Biomaterials 2006; 27: 714–723.
