اثر استرادیول 17- بتا بر بیان ژن آنزیم های استروژنیک و پروژستاژنیک در سلول های گرانولوزا گاوی در شرایط برون تنی

نوع مقاله: مقاله کامل

نویسندگان

1 گروه علوم دامی، دانشگاه علوم کشاورزی و منابع طبیعی ساری

2 گروه علوم دامی، پردیس کشاورزی و منابع طبیعی دانشگاه تهران

چکیده

هدف از مطالعه حاضر بررسی تاثیر استرادیول 17- بتا بر بیان نسبی ژن های گیرنده FSHR) FSH)، گیرنده LHR) LH)و آنزیم های استروژنیک و پروژستاژنیک (Progestagenic) در سلول های گرانولوزای کشت داده شده گاو هلشتاین بود. به طور خلاصه، فولیکول های دو تا پنج میلی متری از تخمدان گاوهای بالغ از کشتارگاه جمع آوری شدند، پس از جدا کردن سلول های گرانولوزا در محیط DMEM/F12 به مدت شش روز در در پلیت هایی 24 تایی کشت داده شدند و به منظور شمارش تعداد سلول ها از رنگ تریپان بلو 4/0 درصد استفاده شد. تیمارها شامل 1) 500 هزار سلول گرانولوزا در ml 500 محیط کشت، 2) 500 هزار سلول گرانولوزا در ml 200 محیط کشت، 3) 500 هزار سلول گرانولوزا در ml 500 محیط کشت و استرادیول 17- بتا و 4) 500 هزار سلول گرانولوزا در ml 200 محیط کشت و استرادیول 17- بتا بودند. برای اندازه گیری استرادیول 17- بتا محیط های کشت در روز شش جمع آوری شدند بر اساس روش رادیوایمونواسی (RIA)، غلظت استرادیول 17- بتا اندازه گیری شد. سلول ها بوسیله ترایزول جمع آوری شدند و تا زمان استخراج RNA در 80- درجه سانتیگراد نگهداری شدند. داده ها با استفاده از نرم افزار JMP مورد تجزیه و تحلیل قرار گرفتند. نتایج نشان داد افزودن استرادیول 17- بتا به محیط کشت سلول های گرانولوزای گاو، از یک سو با افزایش بیان ژن آنزیم های کلیدی در تبدیل اندروژن به استرادیول 17- بتا (آنزیم های استروژنیک) غلظت استرادیول 17- بتا تولید شده به وسیله سلول های گرانولوزا را افزایش داد. از سوی دیگر مسیر ساخت پرژوسترون را با کاهش بیان ژن آنزیم های کلیدی در تبدیل کلسترول به پروژسترون (آنزیم های پروژستاژنیک) مهار کرد و در نهایت سبب کاهش لوتئینه شدن و بهبود سلامت فولیکول شد.

عنوان مقاله [English]

Effect of estradiol 17β on relative gene expression of estrogenic and progestagenic enzymes in cow granulosa cells in vitro

نویسندگان [English]

  • E. Dirandeh 1
  • T. Saberifar 2
  • H. Deldar 1
1 Department of Animal Science, Sari agricultural and Natural Resources University
2 Department of Animal Science, College of Agricultural Science and Natural Resources University of Tehran
چکیده [English]

The objective of this study was to determine the effect of estradiol on mRNA levels encoding FSH receptor (FSHR), LH receptor (LHR), estrogenic and progestagenic enzymes. Briefly, follicles between 2 and 5 mm diameter were dissected from the ovaries of adult cows collected from a local abattoir, cells were collected by rinsing the follicle walls with Dulbecco Modified Eagle medium/F12 (DMEM/F12). A viable cell count was performed in the presence of 0.4 % Trypan Blue and were cultured in 24-well plates for 6 days. Treatments were 1) 500,000 cell/500 ml, 2) 500,000 cell/200 ml, 3) 500,000 cell/500 ml and estradiol 4) 500,000 cell/ 200 ml and estradiol. To measure steroid secretion, the medium was removed for steroid assay on day 6 and measured by RIA. To measure mRNA abundance, cells were collected into Trizol and stored at −80 °C until RNA extraction. All data were analyzed by JMP (SAS). Results showed that addition of estradiol to granulosa cell culture medium was stimulated the E2 synthesis in granulosa cells by increasing key enzymes involved in the conversion of androgens to E217β (estrogenic enzyme) and also inhibited the progestin synthetic pathway by decreasing mRNA level of progestagenic enzyme involved in the conversion of cholesterol to P4 and finally decreasing luteinization and improve follicle healthy or oocyte developmental competence.

1- Bao., B. and Garverick. H.A. (1998). Expression of steroidogenic enzyme and gonadotropin receptor genes in bovine follicles during ovarian follicular waves: a review. Journal of Animal Science. 76. pp: 1903–1921.
2- Bao, B. Garverick, H.A. Smith, G.W. Smith, M.F. Salfen, B.E. and Youngquist, R.S. (1997). Changes in messenger ribonucleic acid encoding luteinizing hormone receptor, cytochrome P450-side chain cleavage, and aromatase are associated with recruitment and selection of bovine ovarian follicles. Biology of Reproduction. 56. pp: 1158–1168.
3- Bao, B. Calder, M.D. Xie, S. Smith, M.F. Salfen, B.E. Youngquist, R.S. and Garverick, H.A. (1998). Expression of steroidogenic acute regulatory protein messenger ribonucleic acid is limited to theca of healthy bovine follicles collected during recruitment, selection, and dominance of follicles of the first follicular wave. Biology of Reproduction. 59. pp: 953–959.
4- Belanger, A. Couture, J. Caron, S. and Roy, R. (1990). Determination of nonconjugated and conjugated steroid levels in plasma and prostate after separation on C-18 columns. Ann N YAcad Sci. 595. pp: 251–9.
5- Drummond, A.E. (2006). The role of steroids in follicular growth. Reproductive Biology and Endocrinology. 4. pp:16-23.
6- Fitzpatrick, S.L. and Richards, J.S. (1991). Regulation of cytochrome P450 aromatase messenger ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology.129. pp:1452–1462.
7- Fitzpatrick, S.L. Carlone,D.L. Robker, R.L. and Richards, J.S. (1997). Expression of aromatase in the ovary: down-regulation of mRNA by the ovulatory luteinizing hormone surge. Steroids. 62. pp: 197–206.
8- Fortune, J.E. (1986). Bovine theca and granulosa cells interact to promote androgen production. Biology of Reproduction. 35. pp: 292–299.
9- Gutierrez, C.G. Campbell, B.K. and Webb, R. (1997). Development of a long-term bovine granulosa cell culture system: induction and maintenance of estradiol production, response to follicle-stimulating hormone, and morphological characteristics. Biology of Reproduction. 56. pp: 608–16.
10-Irving-Rodgers, H.F. Krupa, M.Rodgers, R.J. (2003). Cholesterol side-chain cleavage cytochrome P450 and 3beta-hydroxysteroid dehydrogenase expression and the concentrations of steroid hormones in the follicular fluids of different phenotypes of healthy and atretic bovine ovarian follicles. Biology of Reproduction. 69. pp: 2022–2028.
11- Kolibianakis, E.M. Papanikolaou, E.G. Fatemi, H.M. Devroey, P. (2005). Estrogen and folliculogenesis: is one necessary for the other? Current Opinion in Obstetrics and Gynecology 17. pp: 249–253.
12- Mihm, M. Baker, P.J. Fleming, L.M. Monteiro, A.M. O’Shaughnessy, P.J. (2008). Differentiation of the bovine dominant follicle from the cohort upregulates mRNA expression for new tissue development genes. Reproduction. 135. pp: 253–65.
13- Price, C.A. Carriere, P.D. Gosselin, N. Kohram, H. and Guilbault, L.A. (1999). Effects of superovulation on endogenous LH secretion in cattle, and consequences for embryo production. Theriogenology. 51. pp: 37–46.
14- Raffalli-Mathieu, F. Persson, D. and Mannervik, B. (2007). Differences between bovine and human steroid double-bond isomerase activities of Alphaclass glutathione transferases selectively expressed in steroidogenic tissues. Biochimica et Biophysica Acta. 1770. pp: 130–136.
15- Sahmi, M. Nicola, E.S. Silva, J.M. and Price, C.A. (2004). Expression of 17b- and 3b-hydroxysteroid dehydrogenases and steroidogenic acute regulatory protein in non-luteinizing bovine granulosa cells in vitro. Molecular and Cellular Endocrinology. 223. pp: 43–54.
16- Soumano, K. and Price, C.A. (1997). Ovarian follicular steroidogenic acute regulatory protein, low-density lipoprotein receptor, and cytochrome P450 side-chain cleavage messenger ribonucleic acids in cattle undergoing superovulation. Biology of Reproduction. 56. pp: 516–522.
17- Velerio, P.M. Gustavo, Z. Price, C.A. (2010). Cell plating density alters the ratio of estrogenic to progestagenic enzyme gene expression in cultured granulosa cells. Fertility and Sterility. 93. 6. pp: 2050-2055.
18- Xu, Z. Garverick, H.A. Smith, G.W. Smith, M.F. Hamilton, S.A. and Youngquist, R.S. (1995). Expression of messenger ribonucleic acid encoding cytochrome P450 side-chain cleavage, cytochrome p450 17 alpha-hydroxylase, and cytochrome P450 aromatase in bovine follicles during the first follicular wave. Endocrinology. 136. pp: 981–989.
19- Xuesong, Wen. Dong, Li. Tozer, A.J. Suzanne, M. Docherty, R. Iles, K. (2010). Estradiol, progesterone, testosterone profiles in human follicular fluid and cultured granulosa cells from luteinized pre-ovulatory follicles. Reproductive Biology and Endocrinology. 8:117.
20- Zheng, X. Price, C.A. Tremblay, Y. Lussier, J.G. and Carriere, P.D. (2008). Role of transforming growth factor-b1 in gene expression and activity of estradiol and progesterone-generating enzymes in FSH-stimulated bovine granulosa cells. Reproduction. 136. pp: 447–57.