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. 2021 Mar 19;148(6):dev197459.
doi: 10.1242/dev.197459.

Blocking estrogen-induced AMH expression is crucial for normal follicle formation

Ren Tanimoto  1 , Kiyono Sekii  1 , Kanako Morohaku  2 , Jianzhen Li  1 , David Pépin  3   4 , Yayoi Obata  5
Affiliations

Blocking estrogen-induced AMH expression is crucial for normal follicle formation

Ren Tanimoto et al. Development. .

Abstract

In mammals, primordial follicles assembled in fetuses or during infancy constitute the oocyte resources for life. Exposure to 17beta-estradiol and phytogenic or endocrine-disrupting chemicals during pregnancy and/or the perinatal period leads to the failure of normal follicle formation. However, the mechanisms underlying estrogen-mediated abnormal follicle formation and physiological follicle formation in the presence of endogenous natural estrogen are not well understood. Here, we reveal that estrogen receptor 1, activated by estrogen, binds to the 5' region of the anti-Mullerian hormone (Amh) gene and upregulates its transcription before follicle formation in cultured mouse fetal ovaries. Ectopic expression of AMH protein was observed in pregranulosa cells of these explants. Furthermore, the addition of AMH to the culture medium inhibited normal follicle formation. Conversely, alpha-fetoprotein (AFP) produced in the fetal liver reportedly blocks estrogen action, although its role in follicle formation is unclear. We further demonstrated that the addition of AFP to the medium inhibited ectopic AMH expression via estrogen, leading to successful follicle formation in vitro Collectively, our in vitro experiments suggest that upon estrogen exposure, the integrity of follicle assembly in vivo is ensured by AFP.

Keywords: AMH; Alpha-fetoprotein; Estrogen; Estrogen receptor; Follicle formation; Mouse.

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Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Sequential analysis of oocyte cyst breakdown in the cultured ovaries. (A) Immunostaining images representative of oocyte cyst breakdown in ovaries cultured with basal (upper panels, control) or ICI-containing media (lower panels). DDX4 is an oocyte-specific marker. DDX4+ cells are shown in green, and DAPI was used for counterstaining (magenta). (B) Sequential changes in the percentages of single oocytes in the cultured ovaries from days 7 to 13. Oocytes were counted using 1-µm z-stack fluorescent images of the cultured ovaries. Circles and squares indicate the mean percentage of single oocytes in ovaries cultured with basal (control, n=6) and ICI-containing media (n=5 or 6), respectively. Data are mean±s.d. Statistical significance was analyzed using an unpaired two-tailed Student's t-test. *P<0.05.
Fig. 2.
Fig. 2.
Effects of ESR antagonists on secondary follicle formation in the cultured ovaries. Secondary follicle formation was determined by evaluating the numbers of isolated secondary follicles from the cultured ovaries on day 17. The bars indicate the mean number of isolated secondary follicles from single ovaries cultured with basal (control, black), ICI (antagonist of ESR1 and ESR2, an agonist of GPER1, green), MPP (antagonist of ESR1, red), PHTPP (antagonist of ESR2, yellow) or G-1 (an agonist of GPER1 blue)-containing media. Data are mean±s.d. Multiple comparisons with the control were performed using Dunnett's test. *P<0.05, ***P<0.001.
Fig. 3.
Fig. 3.
Effects of ESR antagonists on Amh expression levels in the cultured ovaries. (A) Amh expression levels in the cultured ovaries on day 7. The bars indicate relative expression levels of Amh in the ovaries of P0 mice (white) and those cultured in basal (control, black), ICI (antagonist of ESR1 and ESR2, an agonist of GPER1, green), MPP (antagonist of ESR1, red), PHTPP (antagonist of ESR2, yellow) or G-1 (an agonist of GPER1, blue)-containing media. Tbp was used as an internal control. Data are mean±s.d. Multiple comparisons with the control were performed by Dunnett's test. ***P<0.001. (B) Plot showing the ratio of the mean number of isolated secondary follicles on day 17 of culture to the mean Amh expression level in the cultured ovaries on day 7. The correlation coefficient was R2=0.973. P<0.01.
Fig. 4.
Fig. 4.
Binding of ESR1 to the Amh 5′ region in the ovaries. White bars and black bars indicate relative Amh amount of ESR1 ChIP to IgG ChIP in ovaries from P2 mice and ovaries cultured in basal medium for 9 days, respectively (left). Greb1, known as estrogen-responsive gene, is used as positive control for ESR1 ChIP (right). Data are mean±s.d. Significant differences were analyzed using an unpaired two-tailed Student's t-test. **P<0.01, ***P<0.001.
Fig. 5.
Fig. 5.
Immunostaining analysis of AMH in cultured ovaries. AMH (green) was detected in ovaries cultured in basal medium from day 7 (control) but not in ovaries from P0 and P2 mice. ICI or MPP incorporation largely reduced premature expression of AMH. Nuclei (magenta) were counterstained with DAPI.
Fig. 6.
Fig. 6.
Effects of AMH on secondary follicle formation in cultured ovaries. (A) Efficiency of secondary follicle isolation from cultured ovaries on day 17. The bars indicate the mean number of isolated secondary follicles from single ovaries cultured with SPS-containing medium with or without AMH. Data are mean±s.d. Multiple comparisons with control ovaries cultured with SPS-containing medium without AMH (control) were performed by Dunnett's test. *P<0.05, ***P<0.001. (B) Immunostaining of follicle basement membrane and oocytes using laminin (gray) and DDX4 (green) antibodies, respectively. The ovary cultured with SPS-containing medium without AMH forms individual follicle basement membrane (right panel). Ovaries cultured with SPS and 500 ng/ml AMH-containing medium (middle panel) showed a similar phenotype to ovaries cultured with basal medium (left panel, control).
Fig. 7.
Fig. 7.
Effects of AFP on secondary follicle formation and Amh expression in cultured ovaries. The left graph indicates the efficiency of secondary follicle isolation from the ovaries cultured in basal medium (control, white bar) and AFP-containing medium (black bar) on day 17. The right graph indicates relative expression levels of Amh in ovaries cultured in basal medium (control, white bar) and AFP-containing medium (black bar) on day 7. Data are mean±s.d. Significant differences were analyzed using an unpaired two-tailed Student's t-test. *P<0.05, ***P<0.001.
Fig. 8.
Fig. 8.
Schematic diagram illustrating normal follicle assembly in vivo. Estrogen circulates in the fetal serum during oocyte cyst breakdown and follicle assembly (Dutta et al., 2014). If estrogen binds to ESRs, AMH is prematurely expressed in pregranulosa cells in the ovaries, which in turn delays oocyte cyst breakdown and causes failure of normal follicle assembly. However, AFP also circulates in the fetal serum at a much higher level than E2. AFP captures E2, thereby inhibiting the ESR signaling pathway and/or modulating estrogen action. When follicle assembly is nearly accomplished, AFP disappears. Once primordial follicles enter the growth phase, E2 produced by growing follicles contributes to various biological processes.
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