نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی کارشناسی ارشد، گروه علوم دامی، دانشکدۀ علوم و مهندسی کشاورزی، پردیس کشاورزی و منابع طبیعی، دانشگاه تهران، کرج

2 استادیار گروه علوم دامی، دانشکدۀ علوم و مهندسی کشاورزی، پردیس کشاورزی و منابع طبیعی، دانشگاه تهران، کرج

3 دانشیار گروه علوم دامی، دانشکدۀ علوم و مهندسی کشاورزی، پردیس کشاورزی و منابع طبیعی، دانشگاه تهران، کرج

4 استادیار گروه دام و طیور، پردیس ابوریحان، دانشگاه تهران، پاکدشت

5 استادیار گروه علوم دامی، دانشکدۀ کشاورزی و منابع طبیعی، دانشگاه اراک، اراک

چکیده

در این تحقیق، اثر لایۀ غذادهندۀ STO بر کشت و تکثیر سلول‏های بنیادی اسپرماتوگونی (SSCs) خروس نابالغ در محیط آزمایشگاهی ارزیابی شد. سلول‏های بیضه، از تعداد 30 قطعه خروس نابالغ نژاد ردآیلندرد (8-4 هفته) جدا و در ظرف‏های کشت چهارخانه‏ای به‏طور جداگانه و در حضور فاکتورهای رشد bFGF و LIF در دو تیمار، سه تکرار، و پنج مشاهده در هر تکرار کشت داده شدند. کلنی‏های SSCs در روز پنجم ظاهر و در روزهای هفتم و دهم ارزیابی شدند. تعداد کلنی‏های ایجادشده، مساحت کلنی، و تعداد سلول در هر کلنی روی لایۀ غذادهنده در مقایسه با کلنی‏های کشت‏شده بدون لایۀ غذادهنده بیشتر بود (05/0≥P). نتایج ارزیابی در روز 10 کشت نشان‏دهندۀ کاهش تعداد کلنی، مساحت کلنی، و تعداد سلول/کلنی در هر دو تیمار به نسبت روز هفت کشت است. ژن C-KIT در سلول‏های تشکیل‏دهندۀ کلنی‏ها بیان نشد که می‏تواند دال بر عدم تمایز و داشتن توان خود‏نوزایی سلول‏های موجود در کلنی‏ها باشد. براساس نتایج تحقیق حاضر، استفاده از لایۀ غذادهندۀ STO در کشت و تکثیر SSCs خروس نابالغ مناسب است و توان این سلول‏ها را در تکثیر و خودنوزایی در مقایسه با کشت در تیمار بدون استفاده از لایۀ غذادهنده به‏طور بهتری حفظ می‏کند.

کلیدواژه‌ها

عنوان مقاله [English]

Applying STO as feeder layer for Rooster Spermatogonial Stem cell culture

نویسندگان [English]

  • Rasoul Karimi 1
  • Malek Shakeri 2
  • Mahdi Zhandi 2
  • Hossien Moravej 3
  • Haniyeh Banikamal 1
  • Abdollah Mohammadi-Sangcheshmeh 4
  • Mahdi Khodaei-Motlagh 5

1 M.Sc. Student, Department of Animal Science, Faculty of Agricultural Science & Engineering, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

2 Assistant Professors, Department of Animal Science, Faculty of Agricultural Science & Engineering, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

3 Associate Professor, Department of Animal Science, Faculty of Agricultural Science & Engineering, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

4 Assistant Professor, Department of Animal and Poultry Science, College of Aburaihan, University of Tehran, Pakdasht, Tehran, Iran

5 Assistant Professor, Department of Animal Science, Faculty of Agriculture and Natural Resources, Arak University, Arak, Iran

چکیده [English]

The objective of this study was to evaluate the effect of the STO feeder layer on prepubertal Rhode Island Red rooster SSCs culture and proliferation in vitro. Testis cells from 30 prepubertal Rhode Island Red chicken (4-8 weeks of age), were individually separated and cultivated in the presence of  bFGF and LIF growth factors on four well plates with two treatments and three replicats and five observations per each. SSCs colonies appeared on the 5th day of culture. The number of SSCs colonies, cells/colony and colony area was measured on days 7 and 10 for both treatments. The result of the colony assay on the 7th day revealed significantly higher colony numbers as well as higher cell number/colony and colony area on the STO surface compared to colonies grown on surfaces without a feeder layer (P≤0.05). In contrast, the results of the colony assay on day 10 had declined for both treatments, as compared to day 7. Also, the C-KIT gene was not expressed which is an indication that colonies might be composed of SSCs. In conclusion, these results indicate that the use of the STO feeder layer influences the SSCs proliferation and maintenance of the prepubertal roosters in short-term culture.  

کلیدواژه‌ها [English]

  • avian spermatogonial stem cells
  • cell colony
  • cell feeder layer
  • cell growth factor
  • chicken short term culture
1 . Aponte PM, Soda T, Teerds KJ, Mizrak SC, Van de Kant HJ and De Rooij DG (2008) Propagation of bovine spermatogonial stem cells in vitro. Reproduction. 136(5): 543-557.
2 . Aponte PM, Van Bragt MP, De Rooij DG and Van Pelt AM (2005) Spermatogonial stem cells: characteristics and experimental possibilities. Apmis,. 113(11‐12): 727-742.
3 . Bichun LI, Wang XY, Zhiqan, Xiao XJ, XU Q, CX Wei FY, Sun HC and Chen GH (2010) Directional differentiation of chicken spermatogonial stem cells in vitro. Cytotherapy. 12: 326-331.
4 . Creemers LB, Den Ouden K, Van Pelt AM and De Rooij DG (2002) Maintenance of adult mouse type A spermatogonia in vitro: influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture. Reproduction. 124(6): 791-799.
5 . Dirami G, Ravindranath N, Pursel V and Dym M (1999) Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM. Biology of reproduction. 61(1): 225-230.
6 . Dobrinski I (2005) Germ cell transplantation and testis tissue xenografting in domestic animals. Animal reproduction science. 89(1): 137-145.
7 . Haselbach M, Wegener J, Decker S, Engelbertz C and Galla HJ (2005) Porcine choroid plexus epithelial cells in culture: regulation of barrier properties and transport processes. Microscopy research and technique. 52(1): 137-152.
8 . Honaramooz A, Behboodi E, Megee SO, Overton SA, Galantino-Homer H, Echelard Y and Dobrinski I (2003) Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats. Biology of reproduction. 69(4): 1260-1264.
9 . Jeyaraj DA, Grossman G and Petrusz P (2003) Dynamics of testicular germ cell apoptosis in normal mice and transgenic mice overexpressing rat androgen-binding protein. Reproductive Biology and Endocrinology. 1(1): 48.
10 . Jung JG, Lee YM, Park TS, Park SH, Lim JM and Han JY (2007) Identification, culture, and characterization of germline stem cell-like cells in chicken testes. Biology of Reproduction. 76(1): 173-182.
11 . Kalthoff K and Pomerai Dd (1996) Analysis of biological development. McGraw-Hill New York.
12 . Kimura T, Van Keymeulen A, Golstein J, Fusco A, Dumont JE and Roger PP (2001) Regulation of thyroid cell proliferation by TSH and other factors: a critical evaluation of in vitro models. Endocrine Reviews. 22(5): 631-656.
13 . Kleinman HK, Philp D and MP Hoffman (2003) Role of the extracellular matrix in morphogenesis. Current Opinion in Biotechnology. 14(5): 526-532.
14 . Liu L, He P, Cai1 K, Zhang Y, Li J, Cao1 F, Ding Z and Zhang N (2010) Lentivirus-Mediated Expression of MxA in Chicken Spermatogonial Stem Cells, Reprod Dom Anim. 45: 131-137.
15 . McClusky LM (2003) A scanning electron microscopic study of germ cell maturation in the reproductive tract of the male soupfin shark (Galeorhinus galeus). Acta Zoologica. 84(1): 69-76.
16 . McLean DJ, Russell LD and Griswold MD (2002) Biological activity and enrichment of spermatogonial stem cells in vitamin A-deficient and hyperthermia-exposed testes from mice based on colonization following germ cell transplantation. Biology of Reproduction. 66(5): 1374-1379.
17 . Mitaka T (1998) The current status of primary hepatocyte culture. International Journal of Experimental Pathology. 79(6): 393-409.
18 . Morena AR, Boitani C, Pesce M, Felici M De and Stefanini M (1996) Isolation of highly purified type A spermatogonia from prepubertal rat testis. Andrology. 17(6): 708-717.
19 . Mozdziak PE and Petitte JN (2004) Status of transgenic chicken models for developmental biology. Developmental dynamics. 229(3): 414-421.
20 . Nagano M, Ryu BY, Brinster CJ, Avarbock MR and Brinster RL (2003) Maintenance of mouse male germ line stem cells in vitro. Biology of Reproduction. 68(6): 2207-2214.
21 . Oatley JM, Reeves JJ and McLean DJ (2004) Biological activity of cryopreserved bovine spermatogonial stem cells during in vitro culture. Biology of Reproduction. 71(3): 942-947.
22 . Schrans-Stassen BH, De Rooij DG and Van Pelt AM (1999) Differential expression of c-kit in mouse undifferentiated and differentiating type A spermatogonia. Endocrinology. 140(12): 5894-5900.
23 . Shinohara T, Orwig KE, Avarbock MR and Brinster RL (2000) Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells. Proceedings of the National Academy of Sciences. 97(15): 8346-8351.
24 . Trefil P, Murray RB, Haifen Y, Jiří H and Jiří K (2010) Restoration of spermatogenesis after transplantation of c-Kit positive testicular cells in the fowl. Theriogenology. 74: 1670-1676.
25 . Tres, LL and Kierszenbaum AL (1983) Viability of rat spermatogenic cells in vitro is facilitated by their coculture with Sertoli cells in serum-free hormone-supplemented medium. Proceedings of the National Academy of Sciences. 80(11): 3377-3381.