Establishment of A Reversibly Inducible Porcine Granulosa Cell Line

Abstract

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.

Characterization and establishment of an immortalized rabbit ovary granulosa cell line for reproductive biology experiments

“…established by researchers, such as human [32], mouse [16], rat [33] and porcine [34], etc. but no one has established rabbits GCs lines.…”

Knockout of the Transducin-Like Enhancer of Split 6 Gene Affects the Proliferation and Cell Cycle Process of Mouse Spermatogonia

“…The Tet-on tetracycline induction system is an expression system based on the addition of Dox to induce the expression of a target gene. The Tet-on induction system has been successfully used to achieve conditional induction expression in a variety of cell populations/lines [14][15][16]. In this paper, we used the Tet-on system and the CRISPR/Cas9 system to edit the Tle6 gene, which realized the controllability of cleavage and the conditional induction of a Tle6 gene knockout in mouse spermatogonia.…”

Establishment and Functional Characterization of Immortalized Rabbit Dermal Papilla Cell Lines

“…primary DPC (passage 3) were transfected with pLVX-IRES-Puro-SV40LT lentiviral expression vector [32] (Yingrun Biotechnology, Changsha, China). The puromycin resistance gene (Puro) was used for cell screening [33]. After 72 hours of transfection, DPCs were inoculated with MSCM containing 2.0 μg/mL puromycin for 7 days, and replaced with MSCM containing 1.0 μg/mL puromycin for 2 weeks.…”