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. 2020 Jan 1;98(1):skz376.
doi: 10.1093/jas/skz376.

Regulation of the transcription factor E2F1 mRNA in ovarian granulosa cells of cattle

Breanne C Morrell  1 , M Chiara Perego  1 , Excel Rio S Maylem  1 , Lingna Zhang  1 , Luis F Schütz  1 , Leon J Spicer  1
Affiliations

Regulation of the transcription factor E2F1 mRNA in ovarian granulosa cells of cattle

Breanne C Morrell et al. J Anim Sci. .

Abstract

The E2F family of transcription factors plays an important role in the control of the cell cycle, cell proliferation, and differentiation, and their role in ovarian function is just emerging. Although some evidence suggests a possible role of E2F1 in ovarian follicular development, what regulates its production in ovarian cells is unknown. Objectives of this study were to determine whether: (i) E2F1 gene expression in granulosa cells (GCs) and theca cells (TCs) change with follicular development and (ii) E2F1 mRNA abundance in TC and GC is hormonally regulated. Using real-time PCR, E2F1 mRNA abundance in GC was 5.5-fold greater (P < 0.05) in small (SM; 1 to 5 mm) than large (LG; >8 mm) follicles, but in TC, E2F1 expression did not differ among follicle sizes. SM-follicle GC had 2.1-fold greater (P < 0.05) E2F1 mRNA than TC. In SM-follicle GC, FGF9 induced a 7.6-fold increase in E2F1 mRNA abundance; however, FGF9 did not affect (P > 0.10) abundance of E2F1 mRNA in LG-follicle TC or GC. Follicle-stimulating hormone (FSH) had no effect (P > 0.10) on E2F1 gene expression in SM- or LG-follicle GC. SM-follicle GC were concomitantly treated with insulin-like growth factor 1 (30 ng/mL), FSH (30 ng/mL), and either 0 or 30 ng/mL of FGF9 with or without 50 µM of an E2F inhibitor (E2Fi; HLM0064741); FGF9 alone increased (P < 0.05) GC numbers, whereas E2Fi alone decreased (P < 0.05) GC numbers, and concomitant treatment of E2Fi with FGF9 blocked (P < 0.05) this stimulatory effect of FGF9. Estradiol production was inhibited (P < 0.05) by FGF9 alone and concomitant treatment of E2Fi with FGF9 attenuated (P < 0.05) this inhibitory effect of FGF9. SM-follicle GC treated with E2Fi decreased (P < 0.05) E2F1 mRNA abundance by 70%. Collectively, our studies show that GC E2F1 mRNA is developmentally and hormonally regulated in cattle. Inhibition of E2F1 reduced FGF9-induced GC proliferation and attenuated FGF9-inhibited estradiol production, indicating that E2F1 may be involved in follicular development in cattle.

Keywords: E2F1 transcription factor; cattle; granulosa cell; ovarian follicle; theca cell.

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Figures

Figure 1.
Figure 1.
Expression of E2F1 in freshly collected granulosa and TCs from SM and LG follicles of experiment 1. Results are normalized to constitutively expressed 18S ribosomal RNA. a,bMeans ± SE (n = 7) without a common letter differ (P < 0.05).
Figure 2.
Figure 2.
Effect of FGF9 (30 ng/mL) on E2F1 mRNA expression GCs from SM follicles (experiment 2). Granulosa cells were serum starved for 24 h and then treated for 12 h with either 0 or 30 ng/mL of FGF9. Results are normalized to constitutively expressed 18S ribosomal RNA and expressed as fold (mean ± SE, n = 6) of control values with no additions. Asterisk indicates mean differs from its respective control mean (P < 0.10).
Figure 3.
Figure 3.
Comparison of the effect of FGF9 (30 ng/mL) on E2F1 mRNA expression in TCs and GCs from LG follicles (experiment 3). Both TC and GC were serum starved for 24 h and then treated for 12 h with either 0 or 30 ng/mL of FGF9. Results are normalized to constitutively expressed 18S ribosomal RNA and expressed as fold (mean ± SE, n = 6) of control values for each cell type with no additions.
Figure 4.
Figure 4.
Comparison of the effect of FSH (30 ng/mL) on E2F1 mRNA expression in GCs from SM and LG follicles ( experiment 4). Cells were treated for 24 h with either 0 or 30 ng/mL of FSH. Results are normalized to constitutively expressed 18S ribosomal RNA and expressed as fold (mean ± SE, n = 6) of control values for each cell type with no additions. Main effect of FSH was not significant (P > 0.10). Control values for SM-follicle GC were 28-fold greater than for control values for LG-follicle GC (data not shown).
Figure 5.
Figure 5.
Effects of FGF9 (30 ng/mL), E2Fi (50 µM), and FGF9 plus E2Fi and on cell proliferation (Panel A), and E2 (Panel B) and P4 (Panel C) production in SM-follicle GCs (experiment 5). Treatments were applied for 48 h in serum-free medium containing testosterone, FSH, and IGF1. a–c Within a panel, means ±SE (n = 9)without a common letter differ (P < 0.05).
Figure 6.
Figure 6.
Effect of an E2Fi (50 µM) on E2F1 and FSHR mRNA expression in SM-follicle GCs (experiment 6). Treatments were applied in serum-free medium for 24 h. Results are normalized to constitutively expressed 18S ribosomal RNA and expressed as fold (mean ± SE, n = 6) of control values with no additions. Asterisk indicates mean differs (P < 0.05) from controls.
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References

    1. Aad P. Y., Echternkamp S. E., Sypherd D. D., Schreiber N. B., and Spicer L. J.. . 2012. The hedgehog system in ovarian follicles of cattle selected for twin ovulations and births: evidence of a link between the IGF and hedgehog systems. Biol. Reprod. 87:79. doi:10.1095/biolreprod.111.096735 - PubMed
    1. Albonico M., Schutz L. F., Caloni F., Cortinovis C., and Spicer L. J.. . 2017. In vitro effects of the Fusarium mycotoxins fumonisin B1 and beauvericin on bovine granulosa cell proliferation and steroid production. Toxicon. 128:38–45. doi:10.1016/j.toxicon.2017.01.019 - PubMed
    1. Berisha B., Sinowatz F., and Schams D.. . 2004. Expression and localization of fibroblast growth factor (FGF) family members during the final growth of bovine ovarian follicles. Mol. Reprod. Dev. 67:162–171. doi:10.1002/mrd.10386 - PubMed
    1. Chamberlain C. S., and Spicer L. J.. . 2001. Hormonal control of ovarian cell production of insulin-like growth factor binding proteins. Mol. Cell. Endocrinol. 182:69–81. doi:10.1016/s0303-7207(01)00541-x - PubMed
    1. DeGregori J., and Johnson D. G.. . 2006. Distinct and overlapping roles for E2F family members in transcription, proliferation and apoptosis. Curr. Mol. Med. 6:739–748. doi:10.2174/1566524010606070739 - PubMed