Abstract
In the ovary, the luteinizing hormone (LH) surge suppresses the proliferation and induces the luteinization of preovulatory granulosa cells (GCs), which is crucial for the survival of terminally-differentiated GCs. Krüppel-like factor 4 (Klf4) has been shown to play a role in regulating the cell cycle and apoptosis in various cell types. The rapid induction of Klf4 expressions by LH was observed in preovulatory GCs. To evaluate whether Klf4 affects GC proliferation and survival, primary rat GCs were isolated from pregnant mare serum gonadotropin-primed Sprague–Dawley rat ovaries and transfected with a Klf4 expression vector or Klf4-specific siRNA, followed by determination of the transcript levels of apoptosis-related and cell cycle-related genes. Cell proliferation, viability, and apoptosis were analyzed by BrdU incorporation, a Cell Counting Kit-8 assay, a bioluminescence caspase 3/7 assay, and flow cytometry. LH treatment increased Klf4 mRNA expression in preovulatory GCs. Transcripts of B-cell lymphoma 2 (Bcl-2) and cell cycle promoters (Cyclin D1 and Cyclin D2) decreased, whereas those of the cell cycle inhibitor, p21, increased. Altering the expression of Klf4 by overexpression or knockdown consistently affected the expression of Bcl-2 and Cyclin D1. In agreement with this, Klf4 overexpression reduced cell viability, increased the fraction of apoptotic cells, and arrested cell cycle progression in G1 phase. We conclude that Klf4 increases the susceptibility of preovulatory GCs to apoptosis by down-regulating Bcl-2, and promotes LH-induced cell cycle exit. It appears to be a key regulator induced by the LH surge that determines the fate of GCs in preovulatory follicles during the luteal transition.
Highlights
Whether the follicles are growing or atretic depends on the balance between anti-apoptotic and pro-apoptotic factors that determine the rate of granulosa cell (GC) apoptosis
We investigated whether Krüppel-like factor 4 (Klf4) is regulated by luteinizing hormone (LH) and whether it is involved in cell cycle arrest and the loss of cell viability during the luteal transition period in rat preovulatory GCs in vitro
To investigate changes in the expression of Klf4 and the genes regulating apoptosis and the cell cycle in preovulatory GCs in the early phase of LH exposure, cells were cultured in the presence of LH (0, 100, 200 ng/mL) for 45 min, and real-time PCR analysis was performed (Figure 1)
Summary
Whether the follicles are growing or atretic depends on the balance between anti-apoptotic and pro-apoptotic factors that determine the rate of granulosa cell (GC) apoptosis. The factors regulating follicular cell survival before and after the luteinizing hormone (LH) surge appear to differ. Highly expressed genes in growing preovulatory follicles are turned off by the LH surge, whereas various different genes that are relevant to luteinization increase dramatically during the periovulatory period [3]. Krüppel-like factor 4 (Klf4) expression was first reported in the epithelial lining of the gut and skin, and its effect on cell cycle arrest and apoptosis is different depending on the cellular context [6,7,8]. We investigated whether Klf is regulated by LH and whether it is involved in cell cycle arrest and the loss of cell viability during the luteal transition period in rat preovulatory GCs in vitro. We found that Klf increases the susceptibility of preovulatory GCs to apoptosis by down-regulating Bcl-2, and promotes LH-induced cell cycle exit
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