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Open AccessArticle

Establishment of A Reversibly Inducible Porcine Granulosa Cell Line

School of Life Science and Engineering, Foshan University, Foshan 528231, China
Department of Animal Biosciences, University of Guelph, Guelph, ON N1G 2W1, Canada
College of Animal Science, South China Agricultural University, Guangzhou 510642, China
Author to whom correspondence should be addressed.
Cells 2020, 9(1), 156;
Received: 24 November 2019 / Revised: 1 January 2020 / Accepted: 6 January 2020 / Published: 8 January 2020
Granulosa cells (GCs) are the key components of ovarian follicles for regulating oocyte maturation. Previous established GC lines have allowed prolonged proliferation, but lost some physiological features owing to long-term immortalization. This study was to establish an induced immortal porcine GC line with reversible proliferation status by the tetracycline inducible (Tet-on) 3G system. Our conditional immortal porcine GCs (CIPGCs) line steadily propagated for at least six months and displayed primary GC morphology when cultured in the presence of 50 ng/mL doxycycline [Dox (+)]. Upon Dox withdrawal [Dox (–)], Large T-antigen expression, reflected by mCherry fluorescence, gradually became undetectable within 48 h, accompanied by less proliferation and size increase. The levels of estradiol and progesterone, and the expression of genes associated with steroid production, such as CYP11A1 (cytochrome P450 family 11), 3β-HSD (3β-hydroxysteroid dehydrogenase), StAR (steroidogenic acute regulatory protein), and CYP19A1 (cytochrome P450 family 19 subfamily a member 1), were all significantly higher in the Dox (–) group than Dox (+) group. The CIPGCs could switch into a proliferative state upon Dox induction. Interestingly, the expression of StAR and CYP19A1 in the CIPGCs (–Dox) was significantly increased by adding porcine follicular fluid (PFF) to mimic an ovary follicle environment. Moreover, PFF priming the CIPGCs in Dox (–) group resulted in similar estradiol production as that of primary GC, and enabled this cell line to respond to gonadotrophins in estradiol production. Collectively, we have established an inducible immortal porcine GC line, which offers a unique and valuable model for future research on the regulation of ovarian functions.
Keywords: granulosa cells; Tet-on 3G; Large T; proliferation; immortalization; steroidogenesis granulosa cells; Tet-on 3G; Large T; proliferation; immortalization; steroidogenesis
MDPI and ACS Style

Bai, Y.; Zhu, C.; Feng, M.; Pan, B.; Zhang, S.; Zhan, X.; Chen, H.; Wang, B.; Li, J. Establishment of A Reversibly Inducible Porcine Granulosa Cell Line. Cells 2020, 9, 156.

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