A Novel Mouse Model for Studying the Effects of Cyp17 Overexpression in a Temporal- and Spatial-Specific Manner

Abstract

Abstract Background: Cyp17 plays a key role in theca cells (TCs) to produce androgens, which, in turn, are converted to estrogens in granulosa cells. Intrinsic alterations in ovarian steroidogenesis contribute to excessive ovarian androgen production that characterizes polycystic ovary disease (PCOS)1,2. Hyperandrogenism has been associated with higher levels of Cyp17 in TCs, and correlate with increased numbers of antral follicles3. While androgen excess is one of the hallmark features of PCOS, its putative role in the follicular development and function remains poorly known. Most efforts have used androgen administration or Cyp19 blockade approach to study how androgens prolong folliculogenesis4. Although some insights have been made, it is not clear if these models accurately address the cascade of effects that follow ovarian hyperandrogenism. Aim: Here, we aim to study the specific effects of hyperandrogenemia on ovarian morphology, follicle function and fertility with a new transgenic (TG) mouse model expressing elevated Cyp17 levels exclusively in TCs. Methods: We generated a breeding line of triple TG mice using a combination of the Tet-dependent expression system and the Cre/LoxP gene control system. Specifically, we used Cyp17 promoter-iCre mice crossed with trans-activator mice (R26-STOP-rtTA-IRES-EGFP transgene, Jackson Lab) and with a responder mouse carrying the TRE-Cyp17 transgene. Cyp17 promoter-iCre mice were used to ensure rtTA/EGFP is expressed specifically in TCs of secondary follicles. After the DNA segment between the two LoxP sites is excised by Cyp17iCre specifically in TCs, the R26-STOP-rtTA gene remains activated in all daughter TCs. Only upon treatment with Doxycycline (DOX) can suppression be relieved and active transcription of TRE-Cyp17 be induced in a dose-dependent manner. Results: Cyp17 mRNA expression levels in TCs of TG mice treated with 20, 100 or 200 mg/Kg DOX compared with corresponding untreated control mice showed a modulation in a dose-dependent manner (P=0.01 ANOVA). Confocal and RNAscope analysis validated (i) the effective combination of the Cyp17iCre/rtTA expression system visualizing the rtTA/EGFP specifically expressed in ovarian TCs and (ii) the DOX-induced increase of Cyp17 expression compared with the WT mice. DOX treated TG females were acyclic, being mostly arrested in diestrus. Analysis of estrous cycle stages revealed that treated TG females spent significantly more time in diestrus than control females (P=0.007, ANOVA). Conclusions: Our new in vivo model is the first that analyzes androgen impact independent of any extraovarian source of androgen, complementing current clinical efforts to study the occurrences of TCs elevated androgen levels in normal and PCOS women. 1 Rosenfield, R. L. et al. Endocr Rev (2016)2 Azziz, R. et al. Nat Rev Dis Primers (2016)3 Comim, F. V., et al. Hum Reprod (2013)4 Stener-Victorin, E. et al. Endocr Rev (2020)