While previous studies indicate that pre-pubertal AHRKO antral follicles have slow growth and reduced estradiol production compared to WT follicles [2–6], it was not known whether these alterations were due to low FSH responsiveness in AHRKO versus WT follicles. To investigate this issue, we used an isolated follicle culture system to compare the direct effects of FSH on pre-pubertal WT and AHRKO antral follicles. Our main findings suggest that AHRKO follicles are less responsive to FSH-induced follicle growth than WT follicles because higher levels of FSH are required for AHRKO follicles to reach the same degree of growth as WT follicles. Surprisingly, the response of WT and AHRKO follicles to FSH in terms of steroid hormone production and steroidogenic regulator expression did not follow the response of follicle growth to FSH. Instead, FSH stimulated production of sex steroid hormones and steroidogenic regulators to a similar or greater degree in AHRKO follicles compared to WT follicles. These data suggest that the AHR may contribute to FSH-stimulated follicle growth, but it may not contribute to the ability of FSH to stimulate steroidogenesis.
Based on lower mRNA expression of Fshr as well as reduced binding sites for FSH in AHRKO ovaries compared to WT ovaries already shown in previous studies [5, 8] and the higher FSH levels needed in our experiments for AHRKO follicles to reach WT growth levels, we initially expected that FSH would not be able to stimulate expression of Fshr to the same degree in AHRKO follicles as it does in WT follicles. However, our data indicate an opposite scenario in which FSH increases expression of Fshr to a greater level in AHRKO follicles compared to WT follicles. Further, our data indicate that the down-regulation of Fshr expression upon rising levels of FSH occurred in WT follicles, but not in AHRKO follicles. Because previous studies indicate that FSH can increase its own receptor, leading to increased ability of follicles to respond to FSH [9, 27–30], our data suggest that the reduced levels of Fshr, and thus, the reduced capacity of AHRKO follicles to respond to FSH are not due to an inability of FSH to stimulate expression of its own receptor.
It is unclear how rising levels of FSH down-regulate expression of the Fshr in WT follicles, but not in AHRKO follicles. Previous studies indicate that low levels of FSH may favor increased Fshr expression in granulosa cells, whereas high levels of FSH suppress Fshr expression in granulosa cells . It is possible that differences in Fshr transcript levels in WT and AHRKO follicles treated with rising levels of FSH may be explained by findings suggesting that the AHR is recruited to the Fshr promoter to transcribe the Fshr in WT, but not AHRKO follicles . As FSH levels increase in WT follicles, the high FSH levels may lead to down-regulation of the Fshr. In AHRKO follicles, however, there is no active AHR; therefore, Fshr transcription may occur independently of the AHR. The AHR has been suggested to be indispensable for proper transcription of Cyp19a1 in the ovary . In our experiments, however, Cyp19a1 as well as Inhba transcription followed a very similar pattern in response to FSH in AHRKO follicles compared to WT follicles, suggesting that FSH regulation of Cyp19a1 and Inhba may not directly require the presence of the AHR. Instead, they may require proper transcription of Fshr.
Our experiments used a minimal concentration of 5 IU/mL FSH, which has been shown to be an essential dose for sustaining in vitro follicle growth [6, 20]. Because the physiological doses of FSH that are required to promote growth in antral follicles are unknown, our use of a range of doses of FSH allowed us to compare FSH responsiveness in WT and AHRKO follicles. Unexpectedly, follicles were still able to grow to some degree when FSH was omitted from the culture. This finding is supported by previous studies that have demonstrated that FSHR knockout mice can develop pre-antral follicles up to the early antral follicle stage, but not further [31–33]. In addition, follicles cultured with no FSH treatment during the antral phase have limited granulosa cell differentiation in terms of their ability to produce estradiol . As shown in our experiments, estradiol production and hormone precursors in follicles cultured with no FSH treatment were lower than those cultured with FSH treatment regardless of genotype. Since our in vitro culture uses a luteinizing hormone (LH)-free culture medium, androgen levels may be the result of constitutive androgen production by the theca cells, which provided sufficient substrate for estradiol production .