Isolation of granulosa cells from follicular fluid; applications in biomedical and molecular biology experiments

Aghadavod, Esmat; Zarghami, Nosratollah; Farzadi, Laya; Zare, Mina; Barzegari, Abolfazl; Movassaghpour, Ali Akbar; Nouri, Mohammad

Isolation of granulosa cells from follicular fluid; applications in biomedical and molecular biology experiments

Authors

¹ Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, I.R,; Women's Reproductive Health Research Center, Tabriz, Iran

² Department of Biochemistry and Clinical Laboratories, Tabriz University of Medical Sciences, Tabriz, Iran

³ Women's Reproductive Health Research Center, Tabriz, Iran

⁴ Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran

⁵ Hemathology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

⁶ Women's Reproductive Health Research Center, Tabriz; Department of Biochemistry and Clinical Laboratories, Tabriz University of Medical Sciences, Tabriz, Iran

Abstract

Background: Recently, a lot of research has been conducted to investigate the molecular mechanisms of the low quality of oocytes with granulosa cells (GCs). GCs are one of the major cell types found in follicular fluid and purification of these cells from the follicular fluid is very important for further studies. Although, there are different techniques of purification, a method for separation of highly-pure and minimally-damaged cells is necessary. In this paper, we presented a novel method for high purification of GCs with a large quantity and high purity.
Materials and Methods: Follicular fluid was collected from patients who referred for in vitro fertilization and GCs in follicular fluid were extracted by Ficoll, Percoll and Red blood cell lysing buffer (RLB) methods. Then purity of extracted GCs was assessed by flow cytometry and morphological properties of GCs were observed by differential interference contrast microscopy. The purity of deoxyribonucleic acid and ribonucleic acid extracts was examined by NanoDrop 1000, pre-restriction fragment length polymorphism and electrophoresis techniques. Quality and quantity of extracting GCs were affected during the cell separation procedures.
Results: Our results showed that each of purification method can affect quality and quantity of extracted cells.
Conclusion: RLB method for extraction of GCs was shown to be a convenient procedure in comparison with Ficoll and Percoll methods.

Keywords

References

1.	de Ziegler D, Streuli I, Gayet V, Frydman N, Bajouh O, Chapron C. Retrieving oocytes from small non-stimulated follicles in polycystic ovary syndrome:In vitro maturation (IVM) is not indicated in the new GnRH antagonist era. Fertil Steril 2012;98:290-3.
2.	Pectasides D, Pectasides E, Psyrri A. Granulosa cell tumor of the ovary. Cancer Treat Rev 2008;34:1-12.
3.	Das M, Djahanbakhch O, Hacihanefioglu B, Saridogan E, Ikram M, Ghali L, et al. Granulosa cell survival and proliferation are altered in polycystic ovary syndrome. Obstet Gynecol Surv 2008;63:579-80.
4.	Levay PF, Huyser C, Fourie FL, Rossouw DJ. The detection of blood contamination in human follicular fluid. J Assist Reprod Genet 1997;14:212-7.
5.	Sifer C, Blanc-Layrac G, Bringuier AF, Porcher R, Madelenat P, Feldmann G, et al. Effects of a Gonadotropin-Releasing Hormone agonist and Follicle Stimulating Hormone on the incidence of apoptosis in human luteinized granulosa cells. Eur Obstet Gynecol Reprod Biol 2003;110:43-8.
6.	McKenzie LJ, Pangas SA, Carson SA, Kovanci E, Cisneros P, Buster JE, et al. Human cumulus granulosa cell gene expression: A predictor of fertilization and embryo selection in women undergoing IVF. Hum Reprod 2004;19:2869-74.
7.	Canipari R. Oocyte–Granulosa cell interactions. Hum Reprod Update 2000;6:279-89.
8.	Attaran M, Frasor J, Mascha E, Radwanska E, Rawlins RG. The relationship of human granulosa-lutein cell proliferative index to follicular diameter and serum estradiol. Obstet Gynecol 1998;91:449-53.
9.	Asem EK, Feng S, Stingley-Salazar SR, Turek JJ, Peter AT, Robinson JP. Basal lamina of avian ovarian follicle: Influence on morphology of granulosa cells in-vitro. Comp Biochem Physiol C Toxicol Pharmacol 2000;125:189-201.
10.	Grøndahl ML, Borup R, Lee YB, Myrhøj V, Meinertz H, Sørensen S. Differences in gene expression of granulosa cells from women undergoing controlled ovarian hyperstimulation with either recombinant follicle-stimulating hormone or highly purified human menopausal gonadotropin. Fertil Steril 2009;91:1820-30.
11.	Catteau-Jonard S, Jamin SP, Leclerc A, Gonzalès J, Dewailly D, di Clemente N. Anti-Mullerian hormone, its receptor, FSH receptor, and androgen receptor genes are overexpressed by granulosa cells from stimulated follicles in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2008;93:4456-61.
12.	Yeo C, Saunders N, Locca D, Flett A, Preston M, Brookman P, et al. Ficoll-Paque versus Lymphoprep: A comparative study of two density gradient media for therapeutic bone marrow mononuclear cell preparations. Regen Med 2009;4:689-96.
13.	Centurione L, Giampietro F, Sancilio S, Piccirilli M, Artese L, Tiboni GM, et al. Morphometric and ultrastructural analysis of human granulosa cells after gonadotrophin-releasing hormone agonist or antagonist. Reprod Biomed Online 2010;20:625-33.
14.	Atashpaz S, Khani S, Barzegari A, Barar J, Vahed SZ, Azarbaijani R, et al. A robust universal method for extraction of genomic DNA from bacterial species. Mikrobiologiia 2010;79:562-6.
15.	Nottola SA, Heyn R, Camboni A, Correr S, Macchiarelli G. Ultrastructural characteristics of human granulosa cells in a coculture system for in vitro fertilization. Microsc Res Tech 2006;69:508-16.
16.	Neubourg DD, Robins A, Fishel S, Gibbon L. Flow cytometric analysis of granulosa cells from follicular fluid after follicular stimulation. Hum Reprod 1996;11:2211-4.
17.	Quinn MC, McGregor SB, Stanton JL, Hessian PA, Gillett WR, Green DP. Purification of granulosa cells from human ovarian follicular fluid using granulosa cell aggregates. Reprod Fertil Dev 2006;18:501-8.
18.	Tan SC, Yiap BC. DNA, RNA, and protein extraction: The past and the present. J Biomed Biotechnol 2009;2009:574398.
19.	Chacon-Cortes D, Haupt LM, Lea RA, Griffiths LR. Comparison of genomic DNA extraction techniques from whole blood samples: A time, cost and quality evaluation study. Mol Biol Rep 2012;39:5961-6.
20.	Wang Q, Wang X. Comparison of methods for DNA extraction from a single chironomid for PCR analysis. Pak J Zool 2012;44:421-6.
21.	Venturoli D, Rippe B. Ficoll and dextran vs. globular proteins as probes for testing glomerular permselectivity: Effects of molecular size, shape, charge, and deformability. Am J Physiol Renal Physiol 2005;288:F605-13.
22.	Motta PM, Nottola SA, Familiari G, Makabe S, Stallone T, Macchiarelli G. Morphodynamics of the follicular-luteal complex during early ovarian development and reproductive life. Int Rev Cyto 2002;223:177-288.
23.	Ferrero H, Delgado-Rosas F, Garcia-Pascual CM, Monterde M, Zimmermann RC, Simón C, et al. Efficiency and purity provided by the existing methods for the isolation of luteinized granulosa cells: A comparative study. Hum Reprod 2012;27:1781-9.

Advanced Biomedical Research