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.
Highlights
Granulosa cells (GCs) play important roles in oocyte development, ovulation, and pregnancy [1].Granulosa cell culture in vitro is a valuable model for studying female reproductive hormone synthesis, follicular development, and fertility [2]
Previous studies have reported that GCs can be immortalized by transfecting genes such as Simian virus 40 Large T-antigen (Large T) [4,5] or telomerase reverse transcriptase (Tert) [6]
The immortalized GC lines from various species have been previously established by expression of Large T and Tert [4,5,8] or spontaneous immortalization [9]
Summary
Granulosa cells (GCs) play important roles in oocyte development, ovulation, and pregnancy [1].Granulosa cell culture in vitro is a valuable model for studying female reproductive hormone synthesis, follicular development, and fertility [2]. Previous studies have reported that GCs can be immortalized by transfecting genes such as Simian virus 40 Large T-antigen (Large T) [4,5] or telomerase reverse transcriptase (Tert) [6]. The immortalized GC lines from various species have been previously established by expression of Large T and Tert [4,5,8] or spontaneous immortalization [9] These GC lines can proliferate in vitro and retain some functional characteristics of primary GCs [10]. A GC line that more closely resembles the primary GCs would be desirable for studying the function and regulation of folliculogenesis [16]
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