Isolation and culture of chicken primordial follicles.

The establishment of a primordial follicle culture system is important for the study of follicular development. Hence, the objective of this study was to isolate chicken primordial follicles and establish culture methods. Ovaries from 2-wk-old chickens were treated with trypsin-EDTA, collagenase II, or collagenase type IA, along with a mechanical isolation technique. Isolated follicles were cultured under different conditions. Results showed a significant difference in the follicular recovery and survival rates among different enzymes and methods used. The maximal follicular yield was obtained by trypsin+EDTA and collagenase II digestion, followed by collagenase type IA digestion. However, the highest follicular viability rate was observed in groups of collagenase type IA digestion and the mechanical isolation method. Enzymatic treatment resulted in higher misshapen oocytes or follicles, though the diameters of the follicles were not significantly changed. In addition, our follicle culture results for different conditions showed maximal survival rates of primordial follicles in alginate hydrogel beads after 12 d of culture. Thus, we successfully established methods for isolating and culturing chicken primordial follicles. The present method will greatly facilitate investigation of the regulation of follicular development.

[1]  J. R. Figueiredo,et al.  Sphingosine 1-phosphate promotes activation of aprine preantral follicle in vitro , 2014 .

[2]  J. Lim,et al.  Development of a serum‐free defined system employing growth factors for preantral follicle culture , 2013, Molecular reproduction and development.

[3]  L. Shea,et al.  Isolated primate primordial follicles require a rigid physical environment to survive and grow in vitro. , 2012, Human reproduction.

[4]  J. Donnez,et al.  Enzymatic isolation of human primordial and primary ovarian follicles with Liberase DH: protocol for application in a clinical setting. , 2011, Fertility and sterility.

[5]  L. Shea,et al.  In vitro grown human ovarian follicles from cancer patients support oocyte growth. , 2009, Human reproduction.

[6]  M. Nagano,et al.  Effects of isolation method and pre-treatment with ethylene glycol or raffinose before vitrification on in vitro viability of mouse preantral follicles. , 2007, Biomedical research.

[7]  Yan Xu,et al.  An efficient isolation method for domestic hen (Gallus domesticus) ovarian primary follicles. , 2006, The Journal of reproduction and development.

[8]  J. Donnez,et al.  Evaluation of Liberase, a purified enzyme blend, for the isolation of human primordial and primary ovarian follicles. , 2006, Human reproduction.

[9]  W. Halfter,et al.  Extracellular Matrices of the Avian Ovarian Follicle , 2004, Journal of Biological Chemistry.

[10]  N. Groome,et al.  Ovarian follicle development in the laying hen is accompanied by divergent changes in inhibin A, inhibin B, activin A and follistatin production in granulosa and theca layers. , 2003, The Journal of endocrinology.

[11]  R. Rumpf,et al.  Zebu (Bos indicus) ovarian preantral follicles: morphological characterization and development of an efficient isolation method. , 2002, Theriogenology.

[12]  M. Skinner,et al.  Leukemia inhibitory factor (LIF) promotes the primordial to primary follicle transition in rat ovaries , 2002, Molecular and Cellular Endocrinology.

[13]  S. Gupta,et al.  Effects of isolation and culture of turkey primary follicular oocytes on morphology and germinal vesicle integrity. , 1998, Theriogenology.

[14]  N. Spears,et al.  The biology and technology of follicular oocyte development in vitro , 1993 .

[15]  M. Skinner,et al.  Platelet-derived growth factor modulates the primordial to primary follicle transition. , 2006, Reproduction.